Polypeptide, Use and Method for Hydrolysing Protein

ABSTRACT

Provided herein is a method for producing a protein hydrolysate using a polypeptide having endopeptidase activity and a polypeptide having carboxypeptidase activity and the use of these enzymes for hydrolysing a protein substrate. Also provided are polypeptides having carboxypeptidase activity and polynucleotides encoding the polypeptides, nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to a method for producing a proteinhydrolysate using a polypeptide having endopeptidase activity and apolypeptide having carboxypeptidase activity and the use of theseenzymes for hydrolysing a protein substrate. In addition the presentinvention relates to polypeptides having carboxypeptidase activity andpolynucleotides encoding the polypeptides. The invention also relates tonucleic acid constructs, vectors, and host cells comprising thepolynucleotides as well as methods of producing and using thepolypeptides.

Description of the Related Art

Protein hydrolysates are used as an additive or ingredient in variousfood products. The advantages of using protein hydrolysates in foodproducts are reduced allergenicity, easier digestion and absorbtion andthereby also faster absorption of the nutrients. Such products can beused in medical nutrition, infant nutrition, health foods, sportsnutritions or can be used for enhancing the protein content of the foodproduct. Also protein hydrolysates are used for enhancing the flavour ofthe food product e.g. by adding umami taste to the product.

Conventionally, protein hydrolysates are produced chemically byhydrolysing protein (e.g. defatted soy flour or wheat gluten) withhydrochloric acid. The hydrolysates are cheap to produce, but thechemical hydrolysis results in byproducts, which are undesirable in foodproducts.

An alternative method for hydrolysing protein is enzymatic hydrolysis,where protein substrate is subjected to peptidases, e.g. as Flavourzyme®(Novozymes) or Alcalase® (Novozymes). The peptidases used forhydrolysing can be either endopeptidases or exopeptidases, where theexo-peptidases are categorised in aminopeptidases and carboxypeptidases.Endoproteases attack proteins and peptides within the molecule.Exoproteases attack from the terminal of the molecule, whereaminopeptidases and carboxypeptidases cleaves off amino acids orpeptides from the protein substrate from the amino-terminal end or thecarboxy-terminal end, respectively.

International patent application WO2016/210395 concerns use ofaminopeptidases for producing protein hydrolysate. EP0946106 describes amethod for producing a protein hydrolysate with a proteolytic enzymemixture comprising only one exo-peptidase.

SUMMARY OF THE INVENTION

The invention provides a method for producing a protein hydrolysatewhich method comprises:

-   -   a. providing an aqueous solution or suspension of a protein        substrate; and    -   b. exposing the aqueous solution or suspension of the protein        substrate to a polypeptide having endopeptidase activity and to        a polypeptide having carboxypeptidase activity, to obtain the        protein hydrolysate;    -   wherein the polypeptide having carboxypeptidase activity is        characterised by having a Pro/ACHA*100 ratio of at least 30.

The present invention provides polypeptides having carboxypeptidaseactivity and polynucleotides encoding the polypeptides.

Accordingly, the present invention relates to polypeptides havingcarboxypeptidase activity selected from the group consisting of:

-   -   a. a polypeptide having at least 60% sequence identity to the        mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO:        6, SEQ ID NO: 8, SEQ ID NO; 10;    -   b. a polypeptide encoded by a polynucleotide having at least 60%        sequence identity to the mature polypeptide coding sequence of        SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID        NO: 9 or the cDNA sequence thereof;    -   c. a variant of the mature polypeptide of SEQ ID NO: 2, SEQ ID        NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 comprising a        substitution, deletion, and/or insertion at one or more        positions; and    -   d. a fragment of the polypeptide of (a), (b) or (c) that has        carboxypeptidase activity.

The invention further concerns a liquid or granulate compositioncomprising the polypeptide of the invention. And the invention concernsa whole broth formulation or cell culture composition comprising thepolypeptide of the invention.

The invention also concerns a polynucleotide encoding the inventivepolypeptide, a nucleoic acid construct or expression vector comprisingthe polynucleotide.

Definitions

ACHA—Average Carboxypeptidase Hydrophobic Activity

Based on the activities measured in Assay II, the ACHA can be calculatedas the the average of the specific activity of the carboxypeptidase onthe following substrates: Z-Ala-Ala-OH, Z-Ala-Val-OH, Z-Ala-Ile-OH,Z-Ala-Leu-OH, Z-Ala-Met-OH, Z-Ala-Phe-OH and Z-Ala-Trp-OH. Z-Ala-Pro-OHis not included in this calculation.

ACLA—Average Carboxypeptidase Activity for Lysine and Arginine

Based on the activities measured in Assay II, the ACLA can be calculatedas the average of the specific activity of the carboxypeptidase on thefollowing two substrates: Z-Ala-Lys-OH, Z-Ala-Arg-OH.

Pro/ACHA*100 Ratio—Pro/Average Carboxypeptidase Hydrofobic Activity(ACHA) Ratio

Based on the activities measured in Assay I and Assay H and thecalculation of ACHA, the Pro/ACHA*100 ratio can be calculated as theactivity on Pro as measured in Assay I divided by ACHA (the average ofthe activity on the hydrophobic amino acids Ala, Val, Ile, Met, Phe, Leuand Trp measured in Assay II) and multiplied with 100,

Carboxypeptidase or Polypeptide Having Carboxypeptidase Activity:

The term “carboxy-peptidase” means a protein or a polypeptide havingcarboxypeptidase activity (3.4.16.X, 3.4.17.X, 3.4.18.X) that catalyzesthe cleavage of the peptide bond at the carboxy-terminal end of theprotein or peptide. For purposes of the present invention,carboxypeptidase activity is determined according to the proceduredescribed in the Assay II, where a polypeptide having carboxypeptidaseactivity is capable of cleaving at least one of the substrates in theassay (e.g. Z-Ala-Val-OH). In one aspect, the polypeptides of thepresent invention have at least 20%, e.g., at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, orat least 100% of the carboxypeptidase activity of the mature polypeptideof SECS ID NO: 2, 4, 6, 8 or 10. The terms “polypeptide havingcarboxypeptidase activity” and “carboxypeptidase” are usedinterchangeably.

Endopeptidase, Endoprotease or Polypeptide Having EndopeptidaseActivity:

The term “endopeptidase” means a protein or a polypeptide havingendopeptidase activity (3.4.19.X, 3.4.21.X, 3.4.22.X, 3.4.23.X,3.4.24.X, 3.4.25.X) that catalyzes the cleavage of the peptide bondswithin the protein or peptide molecule. For purposes of the presentinvention, endopeptidase activity is determined according to theprocedure described in Assay III. The terms “polypeptide havingendopeptidase activity”, “endoprotease” and “endopeptidase” are usedinterchangeably.

Aminopeptidase or Polypeptide Having Aminopeptidase Activity:

The term “aminopeptidase” means a protein or a polypeptide havingaminopeptidase activity (3.4.11.X) that catalyzes the cleavage of thepeptide bond at the amino-terminal end of the protein or peptide. Forpurposes of the present invention, aminopeptidase activity is determinedaccording to the procedure described in the Assay IV. The terms“polypeptide having aminopeptidase activity” and “aminopeptidase” areused interchangeably.

Allelic Variant:

The term “allelic variant” means any of two or more alternative forms ofa gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

cDNA:

The term “cDNA” means a DNA molecule that can be prepared by reversetranscription from a mature, spliced, mRNA molecule obtained from aeukaryotic or prokaryotic cell. cDNA lacks intron sequences that may bepresent in the corresponding genomic DNA. The initial, primary RNAtranscript is a precursor to mRNA that is processed through a series ofsteps, including splicing, before appearing as mature spliced mRNA.

Coding Sequence:

The term “coding sequence” means a polynucleotide, which directlyspecifies the amino acid sequence of a polypeptide. The boundaries ofthe coding sequence are generally determined by an open reading frame,which begins with a start codon such as ATG, GTG, or TTG and ends with astop codon such as TAA, TAG, or TGA. The coding sequence may be agenomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control Sequences:

The term “control sequences” means nucleic acid sequences necessary forexpression of a polynucleotide encoding a mature polypeptide of thepresent invention. Each control sequence may be native (i.e., from thesame gene) or foreign (i.e., from a different gene) to thepolynucleotide encoding the polypeptide or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a polypeptide.

Expression:

The term “expression” includes any step involved in the production of apolypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression Vector:

The term “expression vector” means a linear or circular DNA moleculethat comprises a polynucleotide encoding a polypeptide and is operablylinked to control sequences that provide for its expression.

Fragment:

The term “fragment” means a polypeptide or a catalytic orcarboxypeptidase binding domain having one or more (e.g., several) aminoacids absent from the amino and/or carboxyl terminus of a maturepolypeptide or domain; wherein the fragment has carboxypeptidaseactivity.

Host Cell:

The term “host cell” means any cell type that is susceptible totransformation, transfection, transduction, or the like with a nucleicacid construct or expression vector comprising a polynucleotide of thepresent invention. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication.

Isolated:

The term “isolated” means a substance in a form or environment that doesnot occur in nature. Non-limiting examples of isolated substancesinclude (1) any non-naturally occurring substance, (2) any substanceincluding, but not limited to, any enzyme, variant, nucleic acid,protein, peptide or cofactor, that is at least partially removed fromone or more or all of the naturally occurring constituents with which itis associated in nature; (3) any substance modified by the hand of manrelative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance). An isolated substancemay be present in a fermentation broth sample; e.g. a host cell may begenetically modified to express the polypeptide of the invention. Thefermentation broth from that host cell will comprise the isolatedpolypeptide.

Mature Polypeptide:

The term “mature polypeptide” means a polypeptide in its final formfollowing translation and any post-translational modifications, such asN-terminal processing, C-terminal truncation, glycosylation,phosphorylation, etc. In one aspect, the mature polypeptide is aminoacids 1-426 of SEQ ID NO: 2, amino acids 1-443 of SEQ ID NO: 4, aminoacids 1-444 of SEQ ID NO: 6, amino acids 1-477 SEQ ID NO: 8 or aminoacids 1-553 of SEQ ID NO: 10.

It is known in the art that a host cell may produce a mixture of two ofmore different mature polypeptides (i.e., with a different C-terminaland/or N-terminal amino acid) expressed by the same polynucleotide. Itis also known in the art that different host cells process polypeptidesdifferently, and thus, one host cell expressing a polynucleotide mayproduce a different mature polypeptide (e.g., having a differentC-terminal and/or N-terminal amino acid) as compared to another hostcell expressing the same polynucleotide.

Mature Polypeptide Coding Sequence:

The term “mature polypeptide coding sequence” means a polynucleotidethat encodes a mature polypeptide having carboxypeptidase activity. Inone aspect, the mature polypeptide coding sequence is nucleotides 358 to1687 of SEQ ID NO: 1 and nucleotides 1 to 45 of SEQ ID NO: 1 encode asignal peptide.

Nucleic Acid Construct:

The term “nucleic acid construct” means a nucleic acid molecule, eithersingle- or double-stranded, which is isolated from a naturally occurringgene or is modified to contain segments of nucleic acids in a mannerthat would not otherwise exist in nature or which is synthetic, whichcomprises one or more control sequences.

Operably Linked:

The term “operably linked” means a configuration in which a controlsequence is placed at an appropriate position relative to the codingsequence of a polynucleotide such that the control sequence directsexpression of the coding sequence.

Sequence Identity:

The relatedness between two amino acid sequences or between twonucleotide sequences is described by the parameter “sequence identityForpurposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Variant: The term “variant” means a polypeptide having carboxypeptidaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns a method for producing a proteinhydrolysate comprising:

a. providing an aqueous solution or suspension of a protein substrate;andb. exposing the aqueous solution or suspension of the protein substrateto a polypeptide having endopeptidase activity and polypeptide havingcarboxypeptidase activity to obtain the protein hydrolysate;

-   -   wherein the polypeptide having carboxypeptidase activity is        characterised by having a Pro/ACHA*100 ratio of at least 30.

By the use of two different polypeptides, a polypeptide havingendopeptidase activity and a polypeptide having carboxypeptidaseactivity, the protein substrate is attacked in two different ways. Thepolypeptide having endopeptidase activity attacks the protein substrateby cleaving the peptide bonds within the protein molecule, whereby theprotein substrate is hydrolysed to smaller peptides, which can beattacked by the polypeptide having carboxypeptidase activity. The methodthereby provides protein hydrolysates with a high degree of hydrolysis,which are suitable for use in various products, e.g. food products,cosmetic products, medical products.

Any type of protein substrate can be used for protein hydrolysis.Usually the applications of the protein hydrolysate determines the typeof protein substrate used. The protein substrate can be from animal orvegetable source. In one embodiment the animal protein is selected frommilk protein such as casein or whey proteins.

In one embodiment the protein substrate is a vegetable protein selectedfrom the group consisting of cereal, legumes and oilseed. The proteinsubstrate can be from a cereal such as wheat, barley, oat, rye,triticale, maize, rice, sorghum, buckwheat, quinoa, Chia, millet orfonio. In a preferred embodiment, the protein is gluten. In aparticularly preferred embodiment, the protein is wheat gluten. Anothersource of vegetable protein is legumes, where the protein can be frompeas, beans, lentils or chick peas. Protein from oilseeds can be used inthe inventive method e.g. soy bean, cotton seed, peanut, rape seed,sunflower seed, palm kernel, coconut, corn, safflower seed, sunflowerseed and lin seed. In a preferred embodiment the protein substrate issoy protein. The soy protein can be defatted soy (e.g. flakes or flour),soy protein concentrates and soy isolates. The protein content indefatted soy products accounts for 50% of the dry matter, whereas soyprotein concentrates and soy isolates may contain 70% protein and 90%protein, respectively.

The concentration of the protein substrate in the aqueous solution orsuspension should be in the range of 5-35% depending on which proteinsubstrate that is used.

In one embodiment of the invention the concentration of the proteinsubstrate is in the range of 5-30%, in the range of 5-25%, in the rangeof 5-20%, in the range of 5-15% or in the range of 10-15%.

The concentration of the polypeptides used in the method depends on theamount of protein substrate used. In one embodiment of the invention theconcentration of the polypeptide having carboxypeptidase activity is inthe range of 0.01-4 mg enzyme protein/gram protein substrate. In oneembodiment of the invention the concentration of polypeptide is in therange of 0.01-3.9 mg enzyme protein/gram protein substrate, such as inthe range of 0.1-3.8 mg enzyme protein/gram protein substrate, in therange of 0.1-3.6, in the range of 0.2-3.4, in the range of 0.2-3.2, inthe range of 0.3-3.0, in the range of 0.3-2.8, in the range of 0.4-2.6,in the range of 0.4-2.4, in the range of 0.5-2.2, in the range of0.5-2.0.

In one embodiment of the invention, the concentration of the polypeptidehaving endopeptidase activity is in the range of 0.1-3.0% w/w onprotein, such as is in the range of 0.1-2.8% w/w on protein, such as inthe range of 0.1-2.6% w/w on protein, in the range of 0.1-2.4% w/w onprotein, in the range of 0.1-2.2% w/w on protein, in the range of0.1-2.0% w/w on protein, in the range of 0.1-1.8% w/w on protein, in therange of 0.1-1.6% w/w on protein, in the range of 0.1-1.4% w/w onprotein, in the range of 0.1-1.2% w/w on protein, in the range of0.1-1.0% w/w on protein, in the range of 0.2-1.0% w/w on protein, in therange of 0.3-1.0% w/w on protein, in the range of 0.4-1.0% w/w onprotein or in the range of 0.5-1.0% w/w on pro.

The polypeptide having carboxypeptidase activity used in the inventivemethod can be any polypeptide having carboxypeptiddase activity andwhich has a Pro/ACHA*100 ratio of at least 30. The Pro/ACHA*100 ratio isat least 35, at least at least 40, such as at least 41, at least 42, atleast 45, at least 50, at least 51, at least 50, at least 52, at least53, at least 54, at least 55, at least 56, at least 57, at least 58, atleast 59, at least 60 or at least 61.

Some polypeptide having carboxypeptide activity may have an activity onProline when measured with Assay I of at least 0.15. In one embodimentof the invention the activity on Proline when measured with Assay I isat least at least at least 0.16, at least 0.17, at least 0.18, at least0.19, at least 0.2, at least 0.21, at least 0.22, at least 0.23, atleast 0.24, at least 0.25, at least 0.3, at least 0.35, at least 0.4, atleast 0.45, at least 0.5, at least 0.55, at least 0.6 or at least 0.625.

The polypeptides having carboxypeptidase activity tend to have a higheractivity on hydrophobic amino acids. In one embodiment the polypeptidehaving carboxypeptiddase activity has an average hydrophobic activity ofat least 0.51 when measured in Assay II.

In one embodiment, the polypeptide having carboxypeptidase activity hasan ACHA value of at least 0.52 when measured in Assay II, such as atleast 0.53, at least 0.54, at least 0.56, at least 0.57, at least 0.58or at least 0.59.

Some polypeptides having carboxypeptidase activity may have an an ACLAvalue in the range of 0.16-0.25, such as in the range of 0.17-0.25, inthe range of 0.18-0.25, in the range of 0.19-0.25.

Specific polypeptides having carboxypeptidase activity which may be usedin the inventive method are described in the below.

When producing a protein hydrolysate according to the invention, thetemperature of the aqueous solution/suspension comprising the proteinsubstrate should be within the temperature range where the enymes work,so the temperature should be above 5° C. or above 10° C. In oneembodiment, the temperature is in the range of 10-90° C., such as in therange of 20-80° C., in the range of 30-70° C., in the range of 40-60°C., in the range of 45-55° C. or in the range of 50-60° C.

In the inventive method, the protein substrate is exposed to thepolypeptide having endopeptidase activity and the polypeptide havingcarboxypeptidase activity for at least 4 hours. In one embodiment theprotein substrate is exposed to the polypeptide having endopeptidaseactivity and the polypeptide having carboxypeptidase activity for 3-48hours, such as 4-48 hours, 4-36 hours, 6-32 hours, 8-28 hours, 10-26hours, 12-24 hours, 12-22 hours, 12-20 hours, 12-18 hours, 12-16 hoursor for 12-14 hours.

The pH of the aqueous solution/suspension is in the range of 5-8, suchas in the range of 5.5-6.5 or in the range of 6.0-7.0.

When producing the protein hydrolysate, the aqueous solution/suspensioncomprising the protein substrate should be exposed to the polypeptidehaving endopeptidase activity and the polypeptide havingcarboxypeptidase activity either at the same time, or the proteinsubstrate should be exposed to the polypeptide having endopeptidaseactivity first. The advantage of adding the polypeptide havingendopeptidase activity to the aqueous solution/suspension comprising theprotein substrate first, is that the polypeptide having endopeptidaseactivity can hydrolyse the protein substrate and cut into peptides,which are available for the polypeptide having carboxypeptidase activityto hydrolyse.

The hydrolysis can be stopped when the hydrolysed proteins have thedesired degree of hydrolysis, however by the inventive method proteinhydrolysates with a high degree of hydrolysis can be produced. In oneembodiment, the method is carried out until the obtained proteinhydrolysate has a degree of hydrolysis (% DH) in the range of 40%-70%,such as in the range of 45%-70%, in the range of 50%-70%, in the rangeof 55%-70%, in the range of 60%70%, in the range of 65%-70%.

The polypeptide having endopeptidase activity may be obtained from astrain of Bacillus, preferably Bacillus licheniformis or Bacillussubtilis, a strain of Staphylococcus, preferably Staphylococcus aureus.Or the polypeptide having endopeptidase activity may be obtained fromfungal source, e.g. a strain of Streptomyces, preferably Streptomycesthermovularis or Streptomyces griseus, a strain of Actinomyces species,a strain of Aspergillus, preferably Aspergillus aculeates, Aspergillusawamori, Aspergillus foetidus, Aspergillus nidulans, Aspergillus niger,ox Aspergillus oryzae, or a strain of Trichoderma, preferablyTrichoderma reesei, or Fusarium, preferably Fusarium venenatum. Thepolypeptide having endopeptidase activity may be a substilisin. In someembodiments, the endopeptidase is comprised in the products Alcalase®(Novozymes A/S), Flavourzyme® (Novozymes A/S), Savinase® (NovozymesA/S), Esperase® (Novozymes A/S), Alphalase® (Dupont).

The protein substrate can be hydrolysed to an even higher degree ofhydrolysis by exposing the protein substrate to a polypeptide havingaminopeptidase activity or a fermentation broth supernatant of anAspergillus strain having aminopeptidase activity. Amino peptidase 2described in WO2016/210395 (Dupont Nutrition Biosciences APS) can beused in the present invention, especially aminopeptidase 2 defined inSEQ ID Nos: 1-8 or WO2016/210395 is preferred. Other suitableaminopeptidases are described in WO97/29179 (Gist Brocades BV) andWO2003/102195 (DSM IP Assets BV). The protein substrate may be exposedto the polypeptide having aminopeptidase activity at the same time asbeing exposed to the polypeptide having endopeptidase activity. In oneembodiment of the invention, the protein substrate is exposed to thepolypeptide having endopeptidase activity and then subsequently to thepolypeptide having carboxypeptidase activity and the polypeptide havingaminopeptidase activity. In one embodiment, the protein substrate isexposed to the polypeptide having aminopeptidase activity before,simultaneously or after the protein substrate is exposed to thepolypeptide having carboxypeptidase activity.

The concentration of the polypeptide having aminopeptidase activityshould be in the range of 0.05-4 mg enzyme protein per gram proteinsubstrate. In one embodiment, the concentration of the polypeptidehaving aminopeptidase activity may be 0.05-3.5 mg enzyme protein pergram protein substrate, such as 0.05-3.0 mg enzyme protein per gramprotein substrate, 0.05-2.5 mg enzyme protein per gram proteinsubstrate, 0.05-2.0 mg enzyme protein per gram protein substrate,0.06-2.0 mg enzyme protein per gram protein substrate, 0.07-2.0 mgenzyme protein per gram protein substrate, 0.08-2.0 mg enzyme proteinper gram protein substrate, 0.09-2.0 mg enzyme protein per gram proteinsubstrate or 0.1-2.0 mg enzyme protein per gram protein substrate.

In one embodiment, the invention concerns an isolated polypeptide havingcarboxypeptidase activity, selected from the group consisting of:

a. a polypeptide having [at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10;

b. a polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 [if SEQID NO: 1, 3, 5 does not contain any introns, then end (b) here.Otherwise, insert: or the cDNA sequence thereof];

c. a variant of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, orSEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 comprising a substitution,deletion, and/or insertion at one or more positions; and

d. a fragment of the polypeptide of (a), (b) or, (c), or (d) that hascarboxypeptidase activity.

In one embodiment, the invention concerns a protein hydrolysate producedby the inventive method.

In one embodiment, the invention concerns a protein hydrolysatecomprising free amino acids, wherein the amount of free amino acids isat least 20 g per 100 gram protein, the total amount of Ala, Val, Ile,Leu, Met, Phe and Trp is at least 7 g per 100 g protein and at least 1.0g Pro per 100 g protein.

In one embodiment, the amount of free amino acids is at least 25 g per100 g protein hydrolysate, such as at least 30 g per 100 g hydrolysate,at least 30 g per 100 g hydrolysate, at least 35 g per 100 g hydrolysateor at least 40 g per 100 g hydrolysate.

In one embodiment, the amount of Ala, Val, Ile, Leu, Met, Phe and Trp isat least 13 g per 100 g protein substrate and at least 1.8 g per 100 gprotein hydrolysate.

In one embodiment, the hydrolysate further has a degree of hydrolysation(% dH) in the range of 30%-70%, such as in the range of 45%-70%, in therange of 50%-70%, in the range of 55%-70%, in the range of 60%-70%, inthe range of 65%-70%.

Overview of Sequences Listing

SEQ ID NO: 1 is the sequence of a polynucleotide derived fromPenicillium emersonii.SEQ ID NO: 2 is the amino acid sequence of the polypeptide encoded bySEQ ID NO: 1.SEQ ID NO: 3 is the sequence of a polynucleotide derived fromMyceliophthora heterothallica.SEQ ID NO: 4 is the amino acid sequence of the polypeptide encoded bySEQ ID NO: 3.SEQ ID NO: 5 is the sequence of a polynucleotide derived from Chaetomiumstrumarium.SEQ ID NO: 6 is the amino acid sequence of the polypeptide encoded bySEQ ID NO: 5.SEQ ID NO: 7 is the sequence of a polynucleotide derived fromLasiodiplodia theobromae.SEQ ID NO: 8 is the amino acid sequence of the polypeptide encoded bySEQ ID NO: 7.SEQ ID NO: 9 is the sequence of a polynucleotide derived fromThermoascus aurantiacus.SEQ ID NO: 10 is the amino acid sequence of the polypeptide encoded bySEQ ID NO: 9.SEQ ID NO: 11 is the amino acid sequence of a substilisin protease.SEQ ID NO: 12 is the amino acid sequence of a substilisin protease.SEQ ID NO: 13 is the amino acid sequence of a substilisin protease.SEQ ID NO: 14 is the amino acid sequence of carboxypeptidase CPY.SEQ ID NO: 15 is the amino acid sequence of carboxypeptidase CP1.

Polypeptides Having Carboxypeptidase Activity

In an embodiment, the present invention relates to polypeptides having asequence identity to the mature polypeptide of SEQ ID NO: 2 of at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%, which have carboxypeptidase activity. In one aspect,the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 2.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 2 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 70% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 2.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 2 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 75% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 2.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 2 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 80% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 2.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 2 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 85% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 2.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 2 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 90% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 2.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 2 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 95% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 2.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 2 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 100% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 2.

In an embodiment, the present invention relates to polypeptides having asequence identity to the mature polypeptide of SEQ ID NO: 4 of at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%, which have carboxypeptidase activity. In one aspect,the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 4.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 4 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 70% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 4.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 4 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 75% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 4.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 4 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 80% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 4.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 4 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 85% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 4.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 4 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 90% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 4.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 4 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 95% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 4.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 4 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 100% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 4.

In an embodiment, the present invention relates to polypeptides having asequence identity to the mature polypeptide of SEQ ID NO: 6 of at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%, which have carboxypeptidase activity. In one aspect,the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 6.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 6 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 70% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 6.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 6 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 75% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 6.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 6 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 80% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 6.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 6 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 85% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 6.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 6 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 90% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 6.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 6 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 95% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 6.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 6 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 100% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 6.

In an embodiment, the present invention relates to polypeptides having asequence identity to the mature polypeptide of SEQ ID NO: 8 of at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%, which have carboxypeptidase activity. In one aspect,the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 8.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 8 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 70% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 8.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 8 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 75% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 8.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 8 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 80% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 8.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 8 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 85% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 8.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 8 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 90% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 8.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 8 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 95% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 8.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 8 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 100% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 8.

In an embodiment, the present invention relates to polypeptides having asequence identity to the mature polypeptide of SEQ ID NO: 10 of at least60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%, which have carboxypeptidase activity. In one aspect,the polypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, from the mature polypeptide of SEQ ID NO: 10.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 10 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 70% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 10.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 10 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 75% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 10.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 10 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 80% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 10.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 10 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 85% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 10.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 10 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 90% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 10.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 10 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 95% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 10.

In a particular embodiment, the invention relates to polypeptides havinga sequence identity to the mature polypeptide of SEQ ID NO: 10 of atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, and wherein the polypeptide has atleast at least 100% of the carboxypeptidase activity of the maturepolypeptide of SEQ ID NO: 10.

In an embodiment, the polypeptide has been isolated. A polypeptide ofthe present invention preferably comprises or consists of the amino acidsequence of SEQ ID NO: 2, 4 or 6, 8, 10 or an allelic variant thereof;or is a fragment thereof having carboxypeptidase activity. In anotheraspect, the polypeptide comprises or consists of the mature polypeptideof SEQ ID NO: 2, 4, 6. 8 or 10. In another aspect, the polypeptidecomprises or consists of amino acids-119-426 of SEQ ID NO: 2. In anotheraspect, the polypeptide comprises or consists of amino acids-111-443 ofSEQ ID NO: 4. In another aspect, the polypeptide comprises or consistsof amino acids 110-444 of SEQ ID NO: 6. In another aspect, thepolypeptide comprises or consists of amino acids 1-477 of SEQ ID NO: 8.In another aspect, the polypeptide comprises or consists of aminoacids-7-553 of SEQ ID NO: 10.

The polynucleotide of SEQ ID NO: 1, 3, 5, 7, 9 or a subsequence thereof,as well as the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10 or a fragmentthereof may be used to design nucleic acid probes to identify and cloneDNA encoding polypeptides having carboxypeptidase activity from strainsof different genera or species according to methods well known in theart. In particular, such probes can be used for hybridization with thegenomic DNA or cDNA of a cell of interest, following standard Southernblotting procedures, in order to identify and isolate the correspondinggene therein. Such probes can be considerably shorter than the entiresequence, but should be at least 15, e.g., at least 25, at least 35, orat least 70 nucleotides in length. Preferably, the nucleic acid probe isat least 100 nucleotides in length, e.g., at least 200 nucleotides, atleast 300 nucleotides, at least 400 nucleotides, at least 500nucleotides, at least 600 nucleotides, at least 700 nucleotides, atleast 800 nucleotides, or at least 900 nucleotides in length. Both DNAand RNA probes can be used. The probes are typically labeled fordetecting the corresponding gene (for example, with ³²P, ³H, ³⁵S,biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having carboxypeptidase activity. Genomic or otherDNA from such other strains may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that hybridizes with SEQ ID NO: 1, 3,5, 7, 9 or a subsequence thereof, the carrier material is used in aSouthern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1, 3, 5, 7 or 9; (ii) the mature polypeptide codingsequence of SEQ ID NO: 1, 3, 5, 7 or 9; (iii) the cDNA sequence thereof;(iv) the full-length complement thereof; or (v) a subsequence thereof;under very low to very high stringency conditions. Molecules to whichthe nucleic acid probe hybridizes under these conditions can be detectedusing, for example, X-ray film or any other detection means known in theart.

In another embodiment, the present invention relates to a polypeptidehaving carboxypeptidase activity encoded by a polynucleotide having asequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1, 3, 5, 7 or 9 or the cDNA sequence thereof of at least 60%, e.g.,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%. In a further embodiment, the polypeptide has been isolated.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 2, 4, 6, 8 or 10 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions. In an embodiment, the number of amino acid substitutions,deletions and/or insertions introduced into the mature polypeptide ofSEQ ID NO: 2, 4, 6, 8 or 10 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, or 10. The amino acid changes may be of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of 1-30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to 20-25 residues; or a smallextension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly,

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant moleculesare tested for carboxypeptidase activity to identify amino acid residuesthat are critical to the activity of the molecule. See also, Hilton etal., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzymeor other biological interaction can also be determined by physicalanalysis of structure, as determined by such techniques as nuclearmagnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Carboxypeptidase Activity

A polypeptide having carboxypeptidase activity of the present inventionmay be obtained from microorganisms of any genus. For purposes of thepresent invention, the term “obtained from” as used herein in connectionwith a given source shall mean that the polypeptide encoded by apolynucleotide is produced by the source or by a strain in which thepolynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

In one embodiment, the polypeptide is a Penicillium polypeptide, e.g., apolypeptide obtained from Penicillium emersonii.

In one embodiment, the polypeptide is a Myceliophthora polypeptide,e.g., a polypeptide obtained from Myceliophthora heterothallica.

In one embodiment, the polypeptide is a Chaetomium polypeptide, e.g., apolypeptide obtained from Chaetomium strumarium.

In one embodiment, the polypeptide is a Lasiodiplodia polypeptide, e.g.,a polypeptide obtained from Lasiodiplodia theobromae.

In one embodiment, the polypeptide is a Thermoascus polypeptide, e.g., apolypeptide obtained from Thermoascus aurantiacus.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Nucleic Add Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of the polypeptide. Manipulation of the polynucleotideprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including variant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xyIA and xyIB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isornerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dana (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspatic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and variant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyl), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isornerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus otyzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used,

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding thepolypeptide would be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extra-chromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB(phosphoribosylaminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris an hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMß1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus altitudinis, Bacillusamyloliquefaciens, B. amyloliquefaciens subsp. plantarum, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentos, Bacilluslicheniformis, Bacillus megaterium, Bacillus methylotrophicus, Bacilluspumilus, Bacillus safensis, Bacillus stearothermophilus, Bacillussubtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp, Zooepidemicus cells,

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitifis,Streptomyces coelicolor Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ, Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbial. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbial, Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oornycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteria. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveramyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandere, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergiliusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chtysosporium keratinophilum, Chtysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophile, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiate, Pleurotus eryngii,Thielavia terrestris, Trametes villose, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito of al., 1983, J. Bacterial. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and optionally, (b) recovering thepolypeptide. In one aspect, the cell is a Aspergillus cell. In anotheraspect, the cell is a Aspergillus oryzae cell.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and optionally, (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cells may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides having carboxypeptidase activity. Thesedetection methods include, but are not limited to, use of specificantibodies, formation of an enzyme product, or disappearance of anenzyme substrate. For example, an enzyme assay may be used to determinethe activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, a fermentation broth comprising thepolypeptide is recovered.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, UCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

The invention is further summarized in the following paragraphs:

-   -   1. A method for producing a protein hydrolysate comprising:        -   a. providing an aqueous solution or suspension of a protein            substrate; and        -   b. exposing the aqueous solution or suspension of the            protein substrate to a polypeptide having endopeptidase            activity and to a polypeptide having carboxypeptidase            activity, to obtain the protein hydrolysate;        -   wherein the polypeptide having carboxypeptiddase activity is            characterised by having a Pro/ACHA*100 ratio of at least 30.    -   2. Method according to paragraph 1, wherein the Pro/ACHA*100        ratio is at least 35, at least at least 40, such as at least 41,        at least 42, at least 45, at least 50, at least 51, at least 50,        at least 52, at least 53, at least 54, at least 55, at least 56,        at least 57, at least 58, at least 59, at least 60 or at least        61.    -   3 Method according to any of paragraphs 1-2, wherein the protein        substrate is a vegetable protein or an animal protein.    -   4. Method according to paragraph 3, wherein the vegetable        protein substrate is selected from the group consisting of        cereal, legumes and oilseed.    -   5. Method according to paragraph 4, wherein the vegetable        protein substrate from cereal is selected from the group        comprising wheat, barley, oat, rye, triticale, maize, rice,        sorghum, buckwheat, quinoa, chia, millet or fonio.    -   6. Method according to paragraph 5, wherein the protein        substrate is gluten, preferably wheat gluten.    -   7. Method according to paragraph 4, wherein the vegetable        protein substrate from legumes is selected from the group        consisting of peas, beans, lentils and chick peas.    -   8. Method according to paragraph 4, wherein the vegetable        protein substrate from oilseed is selected from soy bean, cotton        seed, peanut, rape seed, sunflower seed, palm kernel, coconut,        corn, safflower seed, sunflower seed and lin seed.    -   9. Method according to paragraph 8, wherein the protein        substrate is soy protein.    -   10. Method according to paragraph 3, wherein the animal protein        is selected from milk proteins such as casein or whey proteins.    -   11. Method according to any of the preceding paragraphs, wherein        the concentration of the protein substrate in the aqueous        solution or suspension is in the range of 5-35%.    -   12. Method according to paragraph 11, wherein the concentration        of the protein substrate is in the range of 5-30%, in the range        of 5-25%, in the range of 5-20%, in the range of 5-15% or in the        range of 10-15%.    -   13. Method according to any of the preceding paragraphs, wherein        the polypeptide having carboxypeptidase activity has an activity        of at least 0.15 on Proline when measured in Assay 1, such as at        least at least 0.16, at least 0.17, at least 0.18, at least        0.19, at least 0.2, at least 0.21, at least 0.22, at least 0.23,        at least 0.24, at least 0.25, at least 0.3, at least 0.35, at        least 0.4, at least 0.45, at least 0.5, at least 0.55, at least        0.6 or at least 0.625.    -   14. Method according to any of the preceding paragraphs, wherein        the polypeptide having carboxypeptiddase activity has an average        hydrophobic activity of at least 0.51 when measured in Assay II.    -   15. Method according to any of the preceding paragraphs wherein        the polypeptide having carboxypeptidase activity has an ACHA        value of at least 0.52, such as at least 0.53, at least 0.54, at        least 0.56, at least 0.57, at least 0.58 or at least 0.59.    -   16. Method according to any of paragraphs 1-12, wherein the        polypeptide having carboxypeptidase activity has an ACLA value        in the range of 0.15-0.25.    -   17. Method according to paragraph 16, wherein the polypeptide        having carboxypeptidase activity has an ACLA value in the range        of 0.16-0.25, such as in the range of 0.17-0.25, in the range of        0.18-0.25, in the range of 0.19-0.25.    -   18. Method according to any of the preceding paragraphs wherein        the concentration of the polypeptide having endopeptidase        activity is in the range of 0.1-3.0% w/w on protein.    -   19. Method according to paragraph 18, wherein the concentration        is in the range of 0.1-2.8%, such as in the range of 0.1-2.6%,        in the range of 0.1-2.4%, in the range of 0.1-2.2%, in the range        of 0.1-2.0%, in the range of 0.1-1.8%, in the range of 0.1-1.6%,        in the range of 0.1-1.4%, in the range of 0.1-1.2%, in the range        of 0.1-1.0%, in the range of 0.2-1.0%, in the range of 0.3-1.0%,        in the range of 0.4-1.0% or in the range of 0.5-1.0%.    -   20. Method according to any of the preceding paragraphs wherein        the concentration of the polypeptide having carboxypeptidase        activity is in the range of 0.01-4 mg enzyme protein/gram        protein substrate.    -   21. Method according to paragraph 20, wherein the concentration        is in the range of 0.01-3.9 mg enzyme protein/gram protein        substrate, such as in the range of 0.1-3.8 mg enzyme        protein/gram protein substrate, in the range of 0.1-3.6, in the        range of 0.2-3.4, in the range of 0.2-3.2, in the range of        0.3-3.0, in the range of 0.3-2.8, in the range of 0.4-2.6, in        the range of 0.4-2.4, in the range of 0.5-2.2, in the range of        0.5-2.0.    -   22. The method of any of the preceding paragraphs wherein the        protein substrate is exposed to the polypeptide having        endopeptidase activity and the polypeptide having        carboxypeptidase activity for at least 3 hours.    -   23. Method according to paragraph 22, wherein the protein        substrate is exposed to the polypeptide having endopeptidase        activity and the polypeptide having carboxypeptidase activity        for 3-48 hours, such as 4-48 hours, such as 4-36 hours, 6-32        hours, 8-28 hours, 10-26 hours, 12-24 hours, 12-22 hours, 12-20        hours, 12-18 hours, 12-16 hours or for 12-14 hours.    -   24. Method according to any of the preceding paragraphs, wherein        method is carried out at a temperature above 10° C.    -   25. Method according to paragraph 24, wherein the temperature is        in the range of 10-90° C., such as in the range of 20-80° C., in        the range of 30-70° C., in the range of 40-60° C., in the range        of 45-55° C. or in the range of 50-60° C.    -   26. Method according to any of the preceding paragraphs, wherein        the pH of the aqueous solution or the aqueous suspension is in        the range of 5-8, such as in the range of 5.5-6.5 or in the        range of 6.0-7.0.    -   27. Method according to any of the preceding paragraphs wherein        the protein substrate is exposed to polypeptide having        endopeptidase activity and the polypeptide having        carboxypeptidase activity at the same time.    -   28. Method according to any of the preceding paragraphs, wherein        the protein substrate is exposed to the polypeptide having        endopeptidase activity before being exposed to the polypeptide        having carboxypeptidase activity.    -   29. Method according to any of the preceding paragraphs, wherein        the protein substrate is further exposed to a polypeptide having        aminopeptidase activity such as aminopeptidase 2.    -   30. Method according to paragraph 29, wherein the aminopeptidase        is the polypeptide of SECS ID NO: 16.    -   31. Method according to paragraph 29, wherein the protein        substrate is exposed to the polypeptide having aminopeptidase        activity at the same time as being exposed to the polypeptide        having endopeptidase activity.    -   32. Method according to paragraph 29, wherein the protein        substrate is exposed to the polypeptide having aminopeptidase        activity before, simultaneously or after the protein substrate        is exposed to the polypeptide having carboxypeptidase activity.    -   33. Method according to paragraph 29, wherein the concentration        of the polypeptide having aminopeptidase activity is in the        range of 0.05-4 mg enzyme protein per gram protein substrate,        such as 0.05-3.5 mg enzyme protein per gram protein substrate,        0.05-3.0 mg enzyme protein per gram protein substrate, 0.05-2.5        mg enzyme protein per gram protein substrate, 0.05-2.0 mg enzyme        protein per gram protein substrate, 0.06-2.0 mg enzyme protein        per gram protein substrate, 0.07-2.0 mg enzyme protein per gram        protein substrate, 0.08-2.0 mg enzyme protein per gram protein        substrate, 0.09-2.0 mg enzyme protein per gram protein substrate        or 0.1-2.0 mg enzyme protein per gram protein substrate.    -   34. Method according to any of the preceding paragraphs, wherein        the method is carried out until the obtained protein hydrolysate        has a degree of hydrolysis (% DH) in the range of 30%-70%, such        as in the range of 45%-70%, in the range of 50%-70%, in the        range of 55%-70%, in the range of 60%-70%, in the range of        65%-70%.    -   35. Method according to any of the preceding paragraphs, wherein        the polypeptide having endopeptidase activity is a subtilisin.    -   36. Method according to paragraph 35, wherein the substilisin is        selected from SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13.    -   37. Method according to any of the preceding paragraphs, wherein        the polypeptide having carboxypeptidase activity is obtained        from fungal source.    -   38. Method according to paragraph 37, wherein the polypeptide        having carboxypeptidase activity is defined in paragraphs 39-45.    -   39. An isolated polypeptide having carboxypeptidase activity,        selected from the group consisting of:        -   a. a polypeptide having at least 60% sequence identity to            the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ            ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10;        -   b. a polypeptide encoded by a polynucleotide having at least            60% sequence identity to the mature polypeptide coding            sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, SEQ            ID NO: 7, SEQ ID NO: 9 or the cDNA sequence thereof;        -   c. a variant of the mature polypeptide of SEQ ID NO: 2, SEQ            ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10            comprising a substitution, deletion, and/or insertion at one            or more positions; and        -   d. a fragment of the polypeptide of (a), (b) or (c) that has            carboxypeptidase activity.    -   40. Polypeptide according to paragraph 39, having at least 60%,        at least 65%, at least 70%, at least 75%, at least 80%, at least        85%, at least 90%, at least 91%, at least 92%, at least 93%, at        least 94%, at least 95%, at least 96%, at least 97%, at least        98%, at least 99% or 100% sequence identity to the mature        polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID        NO: 8 or SEQ ID NO: 10.    -   41. Polypeptide according to any of paragraphs 39-40, which is        encoded by a polynucleotide having at least 60%, at least 65%,        at least 70%, at least 75%, at least 80%, at least 85%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99% or 100% sequence identity to the mature polypeptide coding        sequence of SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:        7 or SEQ ID NO: 9 or the cDNA sequence thereof.    -   42. Polypeptide according to any of paragraphs 39-41, comprising        or consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ        ID NO: 8 or SEQ ID NO: 10; or the mature polypeptide of SEQ ID        NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO:        10.    -   43. Polypeptide according to paragraph 42, wherein the mature        polypeptide is amino acids 1-426 of SEQ ID NO: 2, amino acids        1-443 of SEQ ID NO: 4, amino acids 1-444 of SEQ ID NO: 6, amino        acids 1-477 SEQ ID NO: 8 or amino acids 1-553 of SEQ ID NO: 10.    -   44. Polypeptide according to any of paragraphs 39-43, which is a        variant of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4,        SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 comprising a        substitution, deletion, and/or insertion at one or more        positions.    -   45. Polypeptide according to paragraph 39, which is a fragment        of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ        ID NO: 10, wherein the fragment has carboxypeptidase activity.    -   46. A liquid or granulate composition comprising the polypeptide        of any of paragraphs 39-45.    -   47. A whole broth formulation or cell culture composition        comprising the polypeptide of any of paragraphs 39-45.    -   48. An polynucleotide encoding the polypeptide of any of        paragraphs 39-45.    -   49. A nucleic acid construct or expression vector comprising the        polynucleotide of paragraph 48 operably linked to one or more        control sequences that direct the production of the polypeptide        in an expression host.    -   50. A recombinant host cell comprising the polynucleotide of        paragraph 49 operably linked to one or more control sequences        that direct the production of the polypeptide.    -   51. A method of producing the polypeptide of any of paragraphs        39-45, comprising cultivating a cell, which in its wild-type        form produces the polypeptide, under conditions conducive for        production of the polypeptide.    -   52. Method according to paragraph 51, further comprising        recovering the polypeptide.    -   53. A method for producing a polypeptide having carboxypeptidase        activity, comprising cultivating the host cell of paragraph 50        under conditions conducive for production of the polypeptide.    -   54. Method according to paragraph 53, further comprising        recovering the polypeptide.    -   55. Use of an polypeptide having endopeptidase activity and a        polypeptide having carboxypeptidase activity for hydrolysing a        protein substrate to obtain a protein hydrolysate, wherein the        polypeptide having carboxypeptiddase activity is characterised        by having a Pro/ACHA*100 ratio of at least 30.    -   56. Use according to paragraph 60, wherein the endopeptidase and        a polypeptide having carboxypeptidase activity is used in a        method according to any of paragraphs 1-38.    -   57. Protein hydrolysate produced by the method according to any        of paragraphs 1-38.    -   58. Protein hydrolysate comprising free amino acids, wherein the        amount of free amino acids is at least 20 g per 100 gram        protein, the total amount of Ala, Val, Ile, Leu, Met, Phe and        Trp is at least 7 g per 100 g protein and at least 1.0 g Pro per        100 g protein.    -   59. Protein hydrolysate according to paragraph 58, wherein the        amount of free amino acids is at least 25 g per 100 g protein        hydrolysate, such as at least 30 g per 100 g hydrolysate, at        least 30 g per 100 g hydrolysate, at least 35 g per 100 g        hydrolysate or at least 40 g per 100 g hydrolysate.    -   60. Protein hydrolysate according to any of paragraphs 58-59,        wherein the amount of Ala, Val, Ile, Leu, Met, Phe and Trp is at        least 13 g per 100 g protein substrate and at least 1.8 g per        100 g protein hydrolysate.    -   61. Protein hydrolysate according to any of paragraphs 58-60,        wherein the hydrolysate further has a degree of hydrolysation (%        dH) in the range of 30%-70%, such as in the range of 45%-70%, in        the range of 50%-70%, in the range of 55%-70%, in the range of        60%-70%, in the range of 65%-70%.

Assays

Assay I

Carboxypeptidase Activity for Proline (Z-Ala-Pro-OH Substrate)

Reagents and Chemicals

Assay buffer: 100 mM succinic acid (Sigma), 50 mM KCl (Sigma), 1 mMCaCl2) (Sigma), 0.01% Triton X-100 (Sigma), pH adjusted to 6.0Substrate: Z-L-alanyl-L-Pro-OH substrate (Z-Ala-Pro-OH, Bachem, C-1185)was dissolved in

DMSO to a concentration of 40 mM.

Procedure

25 μL of assay buffer, 25 μL deionized water, 5 μL of the 7-Ala-Pro-OHsubstrate and 10 μL of enzyme diluted to a concentration of 5 μg/mL wereadded to the wells of a 96 well microtiter plate (MTP). The MTP wasincubated at 55° C. for 30 minutes with shaking at 1000 rpm in anEppendorf Thermomixer comfort. Then the MTP was transferred to a fridgekept at 4° C. for 10 minutes. 100 μL of 2.0 M HEPES pH 10.0 and then 100μL of a 6% solution of ninhydrin (Sigma) dissolved in 100% ethanol wereadded to the wells containing the enzyme and substrate solution. The MTPwas incubated at 80° C. for 5 minutes with moderate shaking (350 rpm).The MTP was then kept at room temperature for 10 minutes and theabsorbance of the sample at 450 nm was then measured.

The activity of the enzyme towards (or on) the Z-Ala-Pro-OH substratewas calculated as the absorption at 450 nm minus the backgroundabsorption of a blank (blank sample with 10 μL assay buffer addedinstead of enzyme solution). Results was rounded off to the seconddecimal digit.

Assay II

Carboxypeptidase Assay

Reagents and Chemicals

Assay buffer: 100 mM succinic acid (Sigma), 50 mM KCl (Sigma), 1 mMCaCl2 (Sigma), 0.01% Triton X-100 (Sigma), pH adjusted to 6.0

Stop reagent: 17.9 g trichloro acetic acid (Sigma), 29.9 g sodiumacetate trihydrate (Sigma) and 19.0 mL concentrated acetic acid (Sigma)were mixed and deionized water was added to a final volume of 500 mL

OPA reagent: 47.6 g disodium tetraborate decahydrate (Sigma) and 12.5 gsodium dodecyl sulfate (Sigma) were dissolved in 1 L deionized water

Substrate working solution: The Z-L-alanyl-L-XXX-OH substrate listed intable 1 were dissolved in DMSO or deionized water to a concentration of40 mM (Z-Ala-XXX-OH, where XXX is one of the 17 amino acid listedbelow).

TABLE 1 Code Name Supplier Code Solvent G Z-Ala-Gly-OH Bachem C-1080DMSO A Z-Ala-Ala-OH Bachem C-1045 DMSO S Z-Ala-Ser-OH Bachem C-1215 DMSOV Z-Ala-Val-OH Bachem C-1245 DMSO L Z-Ala-Leu-OH Bachem C-3155 DMSO IZ-Ala-Ile-OH Bachem C-1130 DMSO M Z-Ala-Met-OH Bachem C-1145 DMSO FZ-Ala-Phe-OH Bachem C-1155 DMSO Y Z-Ala-Tyr-OH Bachem C-1235 DMSO WZ-Ala-Trp-OH Bachem C-1225 DMSO D Z-Ala-Asp-OH Bachem C-1070 DMSO EZ-Ala-Glu-OH Bachem C-1075 DMSO N Z-Ala-Asn-OH Bachem C-1065 DMSO HZ-Ala-His-OH Bachem C-1120 DMSO K Z-Ala-Lys-OH Bachem C-1140 dH2O RZ-Ala-Arg-OH Bachem C-1060 DMSO Q Z-Ala-Gln-OH Santa Cruz sc-475969 dH2OBiotechnology

Procedure

Carboxypeptidase activity can be determined using the substrates listedin Table 1 as follows. 50 μL of assay buffer, 10 μL of enzyme diluted toa concentration of 5 μg/mL, 5 μL of the Z-Ala-XXX-OH substrate wereadded to the wells of a 96 well microtiter plate (MTP). The MTP wasincubated at 55° C. for 30 minutes with shaking at 1000 rpm in anEppendorf Thermomixer comfort. Then the MTP was transferred to a fridgekept at 4° C. for 10 minutes. 100 μL of stop reagent was added to thewells containing the enzyme and substrate solution and the MTP wasshaken for 10 seconds at 1000 rpm to ensure complete mixing of the twosolutions. 80 mg ortho-phtaldialdehyde (OPA, Sigma) were dissolved in 2mL ethanol (Sigma) and 88 mg DL-Dithiothreitol (Sigma) were dissolved in2 mL deionized water. The two solutions were added to 80 mL of OPAreagent and the solutions were stirred at room temperature. After 5minutes mixing the solution volume was adjusted to 100 mL with deionizedwater (OPA solution). 225 μL of the freshly-prepared OPA solution wasadded to the wells of a new MTP. 30 μL supernatant from the MTPcontaining enzymes and substrate was added to the well of the secondMTP. The solution was mixed for 10 seconds at room temperature andabsorbance of the sample at 340 nm was measured after 2 minutes ofaddition of the OPA solution.

The activity of the enzyme for a substrate was calculated as theabsorption at 340 nm minus the background absorption of a blank (blanksample with 10 μL assay buffer added instead of enzyme solution).Results were rounded off to the second decimal digit.

Assay III

Testing for endopeptidase activity

Reagent and chemicals

Substrate: Protazyme OL (Megazyme T-PROL 1000).

Temperature: 30° C.

Assay buffer: 100 mM HEPES (Sigma), 50 mM KCl (Sigma), 1 mM CaCl2(Sigma), 0.01% Triton X-100 (Sigma), pH adjusted to 6.0

Procedure

A Protazyme OL tablet (from Megazyme) was suspended in 2.0 ml 0.01%Triton X-100 by gentle stirring. 500 μL of this suspension and 500 μLassay buffer were mixed in an Eppendorf tube and placed on ice. 20 μLAlcalase sample (diluted in 0.01% Triton X-100) was added. The assay wasinitiated by transferring the Eppendorf tube to an Eppendorfthermomixer, which was set to the assay temperature. The tube wasincubated for 15 minutes on the Eppendorf thermomixer at its highestshaking rate (1400 rpm.). The reaction was stopped by transferring thetube back to the ice bath. Then the tube was centrifuged in an ice coldcentrifuge for a few minutes and 200 μL supernatant was transferred to amicrotiter plate. The absorbance at 650 nm (OD650) was read as a measureof protease activity. A blank sample (20 μL 0.01% Triton X-100 insteadof enzyme) was prepared and was included in the assay. The enzymaticactivity was calculated as OD 650(sample)-OD650(blank).

Assay IV

Testing for aminopeptidase activity

Reagents and chemicals

Assay buffer: 100 mM succinic acid (Sigma), 50 mM KCl (Sigma), 1 mMCaCl2) (Sigma), 0.01% Triton X-100 (Sigma), pH adjusted to 6.0

Substrate listed in table 1B were dissolved in DMSO to a concentrationof 40 mM (H-XXX-pNA, where XXX is one of the 18 amino acid listedbelow).

Procedure

100 μL of assay buffer, 5 μL of the H-XXX-pNA substrate and 10 μL ofenzyme diluted to a concentration of 5 μg/mL were added to the wells ofa 96 well microtiter plate (MTP). The MTP was incubated at 55° C. for 60minutes with shaking at 1000 rpm in an Eppendorf Thermomixer comfort.Then the absorbance of the sample at 405 nm was measured.

The activity of the enzyme towards (or on) the H-XXX-pNA substrate wascalculated as the absorption at 405 nm minus the background absorptionof a blank (blank sample with 10 μL assay buffer added instead of enzymesolution). Results was rounded off to the second decimal digit.

TABLE 1B Code Name Supplier Code Solvent G H-Gly-pNA Bachem L-1280 DMSOA H-Ala-pNA Bachem L-1070 DMSO S H-Ser-pNA TAG Copenhagen N.A. DMSO CH-Cys-pNA TAG Copenhagen N.A. DMSO V H-Val-pNA Bachem L-1440 DMSO LH-Leu-pNA Bachem I-1305 DMSO I H-Ile-pNA Bachem L-1815 DMSO M H-Met-pNABachem L-1320 DMSO P H-Pro-pNA Bachem L-1370 DMSO F H-Phe-pNA BachemL-1355 DMSO Y H-Tyr-pNA TAG Copenhagen N.A. DMSO W H-Trp-pNA TAGCopenhagen N.A. DMSO D H-Asp-pNA Bachem L-1525 DMSO E H-Glu-pNA BachemL-1540 DMSO N H-Asn-pNA TAG Copenhagen N.A. DMSO H H-His-pNA BachemL-1785 DMSO K H-Lys-pNA · 2 HBr Bachem L-1315 DMSO R H-Arg-pNA · 2 HClBachem L-1120 DMSO

N.A.=not available

Assay V

OPA assay:

20 μl of a diluted sample with a protein concentration of 0.06-0.1% wasadded to a microtiterplate followed by 200 μl OPA solution. The platewas shaken and absorbance read immediately at 340 nm. OPA solution wasin 100 ml MQ water: 0.504 g sodium bicarbonate, 0.429 g sodium carbonatedecahydrate, 88 mg dithiothreitol, 1 ml 10% SDS, 80 mg ophthaldialdehyde(OPA) in 2 ml 96% ethanol. A standard curve using 125 mg L-serine in 250ml MQ water and diluted 2, 4, 8, 16, 32, 64 fold was included, and theresponse of the samples calculated by comparing to this.

Calculations:

${{DH}(\%)} = \frac{\begin{matrix}{{Ser}\text{-}{equivalents}\mspace{14mu} {in}\mspace{14mu} {sample}\mspace{14mu} \left( {{mg}\mspace{14mu} {{Ser}/{ml}}} \right)*} \\{{Dilution}\mspace{14mu} {factor}\mspace{14mu} \left( {{ml}/g} \right)*100\%}\end{matrix}}{{Protein}\mspace{14mu} {content}\mspace{14mu} {in}\mspace{14mu} {samples}\mspace{14mu} \left( {{g/100}\mspace{14mu} g} \right)*1000\left( {{mg}/g} \right)}$

HPLC Analysis:

Amino acid content was analysed using a ThermoFischer WPS3000 highpressure liquid chromatography system comprising a quaternary pump, anautosampler, a column oven, and a tuneable fluorescence detector. Priorto the analysis, samples were filtered using 0.22 μm PVDF filters andnorvaline added as an internal standard. Samples were analysed afterautomated pre-column derivatization. 28 μL milli-Q water, 10 μL of 0.4 Mborate buffer pH 10.2, 2 μL sample, 2 μL ortho-phthaldialdehyde 2.5 g/Lin 0.1 M borate buffer pH 10.2 and 2 μL fluorenylmethyl chloroformate0.6 g/L in acetonitrile were collected and mixed by pipetting up anddown in a mixing vial. 100 μL milli-Q water was added followed bymixing, and 10 μL was finally injected for chromatographic analysis on aKinetex 5 μm EVO C18 100 Å LC column (150 mm×4.6 mm) with acorresponding SecurityGuard ULTRA cartridge guard column. Solvents were:A: 20 mM potassium phosphate buffer pH 7.2 and B: 50% methanol, 50%acetonitrile. The pump was set to a constant flow rate of 1 ml/minute,and a linear gradient implemented from 0 to 26 min using 3% solvent B upto 60% solvent B. After 26 min, the solvent composition was changed to3% solvent B and the column equilibrated until 35 min. Columntemperature was 40° C. Primary amino acids were excited at 340 nm andemission wavelength was 460 nm while for the secondary amino acids theexcitation wavelength was 288 nm and the emission wavelength 308 nm.Samples were analysed by comparison to an amino acids standard mix in aconcentration range up to 0.3125 mM. All 20 amino acids were analysedexcept cysteine. Histidine and glycine coeluted.

EXAMPLES

Materials and Methods

Overview of Sequences Listing

SEQ ID NO: 1 is the sequence of a polynucleotide derived fromPenicillium emersonii.SEQ ID NO: 2 is the amino acid sequence of the polypeptide encoded bySEQ ID NO: 1.SEQ ID NO: 3 is the sequence of a polynucleotide derived fromMyceliophthora heterothallica.SEQ ID NO: 4 is the amino acid sequence of the polypeptide encoded bySEQ ID NO: 3.SEQ ID NO: 5 is the sequence of a polynucleotide derived from Chaetomiumstrumarium.SEQ ID NO: 6 is the amino acid sequence of the polypeptide encoded bySEQ ID NO: 5.SEQ ID NO: 7 is the sequence of a polynucleotide derived fromLasiodiplodia theobromae.SEQ ID NO: 8 is the amino acid sequence of the polypeptide encoded bySEQ ID NO: 7.SEQ ID NO: 9 is the sequence of a polynucleotide derived fromThermoascus aurantiacus.SEQ ID NO: 10 is the amino acid sequence of the polypeptide encoded bySEQ ID NO: 9.SEQ ID NO: 11 is the amino acid sequence of a substilisin protease.SEQ ID NO: 12 is the amino acid sequence of a substilisin protease.SEQ ID NO: 13 is the amino acid sequence of a substilisin protease.SEQ ID NO: 14 is the amino acid sequence of Carboxypeptidase CPYSEQ ID NO: 15 is the amino acid sequence of Carboxypeptidase CP1SEQ ID NO: 16 is the amino acid sequence of an amino peptidase.

Example 1

Strain

A fungal strain was isolated from a compost sample collected from Yunnanprovince, China by the dilution plate method with FDA medium at 45° C.It was then purified by transferring a single conidium onto a YG agarplate. The strain was identified as Penicillium emersonii, based on bothmorphological characteristics and ITS rDNA sequence.

Media

PDA medium was composed of 39 grams of potato dextrose agar anddeionized water to 1 liter.

YG agar plates were composed of 5.0 g of yeast extract, 10.0 g ofglucose, 20.0 g of agar, and deionized water to 1 liter.

YPG medium contained 0.4% of yeast extract, 0.1% of KH₂PO₄, 0.05% ofMgSO₄.7H₂O, 1.5% glucose in deionized water.

YPM medium contained 1% yeast extract, 2% of peptone, and 2% of maltosein deionized water.

Genomic DNA Extraction of Penicillium emersonii

Penicillium emersonii strain was inoculated onto a PDA plate andincubated for 3 days at 45° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 nil of YPGmedium. The flasks were incubated for 3 days at 45° C. with shaking at160 rpm. The mycelia were collected by filtration through MIRACLOTH®(Calbiochem, La Jolla, Calif., USA) and frozen in liquid nitrogen.Frozen mycelia were ground, by mortar and pestle, to a fine powder, andgenomic DNA was isolated using an Andybio Large-Scale Column FungalDNAout Kit (Bioserver Inc., BeiJing, China).

Genome Sequencing, Assembly and Annotation

The extracted genomic DNA samples were delivered to Beijing GenomeInstitute (BGI, Shenzhen, China) for genome sequencing using anILLUMINA® GA2 System (Illumina, Inc., San Diego, Calif., USA). The rawreads were assembled at BGI using program SOAPdenovo (Li et al., 2010,Genome Research 20(2): 265-72). The assembled sequences were analyzedusing standard bioinformatics methods for gene finding and functionalprediction. GeneID (Parra et al., 2000, Genome Research 10(4): 511-515)was used for gene prediction. Blastall version 2.2.10 (Altschul et al.,1990, J. Mol. Biol. 215 (3): 403-410, National Center for BiotechnologyInformation (NCBI), Bethesda, Md., USA) and HMMER version 2.1.1(National Center for Biotechnology Information (NCBI), Bethesda, Md.,USA) were used to predict function based on structural homology. Thecarboxypeptidase gene, S10_Pe1, was identified directly by analysis ofthe Blast results. The Agene program (Munch and Krogh, 2006, BMCBioinformatics 7: 263) and SignalP program (Nielsen et al., 1997,Protein Engineering 10: 1-6) were used to identify start codons. TheSignalP program was further used to predict signal peptides. Pepstats(Rice et al., 2000, Trends Genet. 16(6): 276-277) was used to predictthe isoelectric points and molecular weights of the deduced amino acidsequences.

Cloning of the Carboxypeptidase Gene of Penicillium emersonii fromGenomic DNA

The expression vector pCaHj505 was used for gene cloning. It containedthe TAKA-amylase promoter derived from Aspergillus oryzae and theAspergillus niger glucoamylase terminator elements. Furthermore pCaHj505had pUC18 derived sequences for selection and propagation in E. coli,and an amdS gene, which encoded an acetoamidase gene derived fromAspergillus nidulans for selection of an amds⁺ Aspergillus transformant.

The carboxypeptidase gene, S10_Pe1, SEQ ID NO: 1 for the genomic DNAsequence and SEQ ID NO: 2 for the deduced amino acid sequence, wasselected for expression cloning.

Based on the DNA information obtained from genome sequencing,oligonucleotide primers, shown below, were designed to amplify thecoding sequence of the carboxypeptdase genes from the genomic DNA ofPenicillium emersonii NN051602. The primers were synthesized byInvitrogen, Beijing, China.

Primer1: [SEQ ID NO: 17] 5′ ACACAACTGGGGATC CACC atgagagttcttcctgcgac 3′Primer2: [SEQ ID NO: 18] 5′ CCCTCTAGATCTCGAG gaacgcgacacgcttctca 3′

Lowercase characters of the forward primer represent the coding regionof the gene and lowercase characters of the reverse primer represent theflanking region of the gene, while captalized characters represent aregion homologous to insertion sites of pCaHj505 (WO2013029496). The 4underlined characters ahead of the coding sequence in the forward primerrepresent the Kozak sequence as the initiation of translation process.

Ten picomoles of the forward and reverse primers above, primer1 andprimer2, were used in a PCR reaction for amplification of thePenicillium emersonii carboxypeptidase gene S10_Pe1. The PCR reactionwas composed of 2 μl of genomic DNA of Penicillium emersonii NN051602,10 μl of 5× Phusion® HF Buffer (Finnzymes Oy, Espoo, Finland), 1.5 μl ofDMSO, 1.5 ul of 2.5 mM each of dATP, dTTP, dGTP, and dCTP, and 0.6 unitof PHUSION® High-Fidelity DNA Polymerase (Finnzymes Oy, Espoo, Finland)in a final volume of 50 μl. The amplification was performed using aPeltier Thermal Cycler (MJ Research Inc., South San Francisco, Calif.,USA) programmed for denaturing at 98° C. for 1 minute; 7 cycles ofdenaturing each at 98° C. for 30 seconds, annealing at 65° C. for 30seconds with 1° C. decrease per cycle, and elongation at 72° C. for 2minutes; 25 cycles each at 94° C. for 30 seconds, 60° C. for 30 seconds,and 72° C. for 2 minutes; and a final extension at 72° C. for 5 minutes.The heat block then went to a 4° C. soak cycle.

The PCR product was isolated by 1.0% agarose gel electrophoresis using90 mM Trisborate and 1 mM EDTA (TBE) buffer where a single product bandof approximately 1.8 kb from the reaction was visualized under UV light.The PCR product was then purified from solution by using an Illustra™GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare,Buckinghamshire, UK) according to the manufacturers instructions.

Plasmid pCaHj505 was digested with Barn HI and Xho I, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

In-Fusion® HD Cloning Kit (Clontech Laboratories, Inc., Mountain View,Calif., USA) was used to clone the PCR fragment directly into theexpression vector pCaHj505, without the need for restriction digestion.

The purified PCR fragment and the digested vector were ligated togetherusing the In-Fusion® HD Cloning Kit according to the manufacturer'sinstructions resulting in plasmid p505-S10_Pe1 (FIG. 1), in which thetranscription of the carboxypeptidase polypeptide coding sequence wasunder the control of an Aspergillus oryzae alpha-amylase gene promoter.In brief, 0.8 ul of 30 ng/ul of pCaHj505, digested with Barn HI and XhoI, and 3.2 ul of the purified PCR fragment containing ˜60 ng of thePenicillium emersonii carboxypeptidase gene PCR fragment were added to 1ul of 5× In-Fusion® HD Enzyme Premix. The reaction was incubated at 50°C. for 15 minutes. The ligation reaction was used to transform E. coliTOP10 competent cells (TIANGEN Biotech Co. Ltd., Beijing, China). E.coli transformants containing an expression construct were detected bycolony PCR. Colony PCR is a method for quick screening of plasmidinserts directly from E. coli colonies. Briefly, a single colony wastransferred to a premixed PCR solution in a PCR tube, including PCRbuffer, MgCl₂, dNTPs, Taq DNA polymerase and primer pairs from which thePCR fragment was generated. Several colonies were screened. After thePCR, reactions were analyzed by 1.0% agarose gel electrophoresis usingTBE buffer. Plasmid DNA was prepared using a QIAPREP® Spin Miniprep Kit(QIAGEN GmbH, Hilden, Germany) from the colony showing an insert withthe expected size. The carboxypeptidase gene coding sequence inserted inp505-S10_Pe1 was confirmed by DNA sequencing using 3730XL DNA Analyzers(Applied Biosystems Inc, Foster City, Calif., USA).

Expression of the Penicillium emersonii Carboxypeptidase Gene inAspergillus oryzae

Aspergillus oryzae strain MT3568 was used for heterologous expression ofthe gene encoding the Penicillium emersonii carboxypeptidase gene. A.oryzae MT3568 is an amdS (acetamidase) disrupted derivative of A. oryzaeJaL355 (WO02/40694) in which pyrG auxotrophy was restored by disruptingthe A. oryzae acetamidase (amdS) gene with the pyrG gene.

Protoplasts were prepared according to the method described as“Transformation of Aspergillus Expression Host” in Example 2 ofUS20140179588 A1. Three μg of p505-S10_Pe1 were used to transformAspergillus oryzae MT3568.

The transformation of Aspergillus oryzae MT3568 with p505-S10_Pe1yielded about 10 transformants. Four transformants were isolated toplate for resolution and were then inoculated separately into 3 ml ofYPM medium in 24-well plate and incubated at 30° C., 150 rpm. After 3days incubation, 20 μl of supernatant from each culture were analyzed onNUPAGE® NOVEX® 4-12% Bis-Tris Gel with 50 mM MES (InvitrogenCorporation, Carlsbad, Calif., USA) according to the manufacturer'sinstructions. The resulting gel was stained with INSTANTBLUE™ (ExpedeonLtd., Babraham Cambridge, UK). SDS-PAGE profiles of the cultures showedthat the majority of the transformants had a smear band of approximately60 kDa. The expression strain was designated as O82P5E.

Fermentation of Aspergillus oryzae Expression Strain O82P5E

Two slants of the expression strain O82P5E, was washed with 10 ml of YPMand inoculated into 18 2-liter flasks each containing 400 ml of YPMmedium, shaking at 30 C, 80 rpm. The culture was harvested on day 3 andfiltered using a 0.45 μm DURAPORE Membrane (Millipore, Bedford, Mass.,USA).

Example 2

Purification of Recombinant Carboxypeptidase by Hydrophobic InteractionChromatography (HIC)

The culture broth harvested in example 1 was precipitated with ammoniumsulfate (80% saturated). Precipitates were re-dissolved in 200 ml of 20mM phosphate-buffered saline (PBS) pH 6.0, and ammonium sulfate wasreplenished to get final concentration 1.2 M. Crude protein solution wasfiltered through a 0.45 μm filter, and then applied to a 50 mlself-packed Phenyl Sepharose 6 Fast Flow (low sub) column (GEHealthcare, Buckinghamshire, UK) equilibrated with 20 mM PBS pH 6.0 and1.2 M ammonium sulfate buffer. Proteins were eluted with a linear 1.2M-0 M ammonium sulfate gradient. Fractions were analyzed by SDS-PAGEusing a Mini-PROTEAN TGX Stain-Free 4-15% Precast Gel (Bio-RadLaboratories, CA, United States). Carboxypeptidase activities offractions were assessed by halo zone assay on skim-milk agarose plate atpH 5.0, 50° C. Fractions were pooled containing recombinant proteinbands and showing positive activities. Then the pooled solution wasconcentrated by ultrafiltration.

Example 3

Cloning and Expression of a S10 Peptidase from Myceliophthoraheterothallica Gene

A fungal strain was isolated and based on both morphological andmolecular characterization (ITS sequencing) classified as Myceliophthoraheterothallica. The Myceliophthora heterothallica strain was annotatedas Myceliophthora heterothallica CBS 202.75 strain and fully genomesequenced. The genomic DNA sequence of a S10 peptidase polypeptideencoding sequence was identified in the genome of Myceliophthoraheterothallica CBS 202.75 strain and the genomic DNA sequence anddeduced amino acid sequence are shown in SEQ ID NO: 3 and SEQ ID NO: 4,respectively. The genomic DNA sequence of 1720 nucleotides contains 1intron of 55 bp (nucleotides 503 to 557). The genomic DNA fragmentencodes a polypeptide of 554 amino acids.

Expression Vector

The Aspergillus expression vector pDau109 (WO 2005/042735) consists ofan expression cassette based on the partly duplicated Aspergillus nigerneutral amylase II (NA2) promoter fused to the Aspergillus nidulanstriose phosphate isomerase non translated leader sequence (Pna2/tpl) andthe Aspergillus niger amyloglycosidase terminator (Tamg). Also presenton the vector is the Aspergillus selective marker amdS from Aspergillusnidulans enabling growth on acetamide as sole nitrogen source and theamplicillin resistance gene (beta lactamase) allowing for facileselection for positive recombinant E. coli clones using commerciallyavailable and highly competent strains on commonly used LB ampicillinplates. pDau109 contains a multiple cloning site situated between thepromoter region and terminator, allowing for insertion of the gene ofinterest in front of the promoter region.

Expression Cloning

The gene encoding the Myceliophthora heterothallica CBS 202.75 S10peptidase (SEQ ID NO: 3) was PCR amplified from genomic DNA isolatedfrom Myceliophthora heterothallica CBS 202.75 strain. The PCR productencoding the Myceliophthora heterothallica CBS 202.75 S10 peptidase (SEQID NO: 3) was cloned into the pDau109 Aspergillus expression vectorusing the unique restriction sites BamHI and HindIII and transformedinto E. coli (Top10, Invitrogen). Expression plasmids containing theinsert were purified from the E. coli transformants, and sequenced withvector primers and gene specific primers in order to determine arepresentative plasmid expression clone that was free of PCR errors. Theplasmid expression clone was transformed into A. oryzae and arecombinant A. oryzae clone containing the integrated expressionconstruct were grown in liquid culture. Expression of the Myceliophthoraheterothallica CBS 202.75 S10 peptidase was verified by SDS-page. Theenzyme containing supernatant was sterile filtered before purification.

Example 4

Purification Assay:

-   -   Substrate: Z-Ala-Phe-OH(Sachem C-1155).    -   Temperature: 37° C.    -   Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES,        100 mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100, pH 6.0.

100 μl Z-Ala-Phe-OH substrate (50 mg dissolved in 1.0 ml DMSO andfurther diluted 25× in 0.01% Triton X-100) was mixed with 1541 Assaybuffer in an Eppendorf tube and placed on ice. 50 μl peptidase sample(diluted in 0.01% Triton X-100) was added. The assay was initiated bytransferring the Eppendorf tube to an Eppendorf thermomixer, which wasset to 37° C. The tube was incubated for 15 minutes on the Eppendorfthermomixer at its highest shaking rate (1400 rpm.). The tube was thentransferred back to the ice bath and when the tube had cooled, 500 μlStop reagent (17.9 g TCA+29.9 g Na-acetate trihydrate+19.0 ml conc.CH₃COOH and deionised water ad 500 ml) was added and the tube wasvortexed and left for 15 min at room temperature (to ensure completeprecipitation). The tube was centrifuged (15000×g, 3 min, room temp), 30μl supernatant was transferred to a microtiter plate and 225 μl freshlyprepared OPA-reagent (3.81 g disodium tetraborate and 1.00 g SDS weredissolved in approx. 80 ml deionised water—just before use 80 mgortho-phtaldialdehyde dissolved in 2 ml ethanol was added and then 1.0ml 10% (w/v) DIE and finally the volume was adjusted ad 100 ml withdeionised water) was added. After 2 minutes, A₃₄₀ was read in a MTPreader. The A₃₄₀ measurement relative to an enzyme blind (50 μl 0.01%Triton X-100 instead of peptidase sample) was a measure ofcarboxypeptidase activity.

Purification of the 510 Carboxypeptidase from Myceliophthoraheterothallica:

(The S10 Carboxypeptidase was Expressed in A. oryzae.)

The culture broth was centrifuged (20000×g, 20 min) and the supernatantwas carefully decanted from the precipitate. The supernatant wasfiltered through a Nalgene 0.2 μm filtration unit in order to remove therest of the Aspergillus host cells. The 0.2 μm filtrate was transferredto 20 mM MES/NaOH, 0.5 mM CaCl₂, pH 6.0 on a G25 sephadex column (fromGE Healthcare). The G25 sephadex transferred carboxypeptidase wasapplied to a Q-sepharose FF column (from GE Healthcare) equilibrated in20 mM MES/NaOH, pH 6.0. After washing the column extensively with theequilibration buffer, the S10 carboxypeptidase was eluted with a lineargradient between the equilibration buffer and 20 mM MES/NaOH, 5 mMCaCl₂, 500 mM NaCl, pH 6.0 over five column volumes. The elutedfractions were analysed for carboxypeptidase activity and activefractions were further analysed by SDS-PAGE. Fractions with one band atapprox. 50 kDa were pooled as the purified product and was used forfurther characterization.

Characteristics for the Purified S10 Carboxypeptidase fromMyceliophthora heterothallica:

The relative molecular weight as determined by SDS-PAGE was approx.Mr=50 kDa.

The major N-terminal sequence determined by EDMAN degradation was:TVDPSKL (60%). Two minor N-terminal sequences were also determined:KTVDPSK (20%) and VKTVDPS (20%) suggesting that that the N-termini weresomewhat ragged.

The S10 carboxypeptidase from Myceliophthora heterothallica wasglycosylated and therefore the purified carboxypeptidase was treatedwith Endo H before Intact MS analysis. The measured peak pattern couldbe assigned to the mature sequence.

Example 5

Cloning and expression of a SIC peptidase from Chaetomium strumariumGene

A fungal strain was isolated and based on both morphological andmolecular characterization (ITS sequencing) classified as Chaetomiumstrumarium. The Chaetomium strumarium strain was annotated as Chaetomiumstrumarium strain and fully genome sequenced. The genomic DNA sequenceof a S10 peptidase polypeptide encoding sequence was identified in thegenome of Chaetomium strumarium strain and the genomic DNA sequence anddeduced amino acid sequence are shown in SEQ ID NO: 5 and SEQ ID NO: 6,respectively. The genomic DNA sequence of 1722 nucleotides contains 1intron of 57 bp (nucleotides 503 to 559). The genomic DNA fragmentencodes a polypeptide of 554 amino acids.

Expression Vector

The Aspergillus expression vector pDau109 (WO 2005/042735) consists ofan expression cassette based on the partly duplicated Aspergillus nigerneutral amylase II (NA2) promoter fused to the Aspergillus nidulanstriose phosphate isomerase non translated leader sequence (Pna2/tpl) andthe Aspergillus niger amyloglycosidase terminator (Tamg). Also presenton the vector is the Aspergillus selective marker amdS from Aspergillusnidulans enabling growth on acetamide as sole nitrogen source and theamplicillin resistance gene (beta lactamase) allowing for facileselection for positive recombinant E. coli clones using commerciallyavailable and highly competent strains on commonly used LB ampicillinplates. pDau109 contains a multiple cloning site situated between thepromoter region and terminator, allowing for insertion of the gene ofinterest in front of the promoter region.

Expression Cloning

The gene encoding the Chaetomium strumarium S10 peptidase (SEQ ID NO: 5)was PCR amplified from genomic DNA isolated from Chaetomium strumariumstrain. The PCR product encoding the Chaetomium strumarium S10 peptidase(SEQ ID NO: 5) was cloned into the pDau109 Aspergillus expression vectorusing the unique restriction sites BamHI and HindIII and transformedinto E. coli (Top10, Invitrogen). Expression plasmids containing theinsert were purified from the E. coli transformants, and sequenced withvector primers and gene specific primers in order to determine arepresentative plasmid expression clone that was free of PCR errors. Theplasmid expression clone was transformed into A. oryzae and arecombinant A. oryzae clone containing the integrated expressionconstruct were grown in liquid culture. Expression of the Chaetomiumstrumarium S10 peptidase was verified by SDS-page. The enzyme containingsupernatant was sterile filtered before purification.

Example 6

Strains

The strain Lasiodiplodia theobromae was isolated from soil, Yunnan,China in 1999.

Cloning of S10 Carboxypeptidase from Lasiodiplodia theobromae

The carboxypeptidase with nucleotide sequence SEQ ID NO: 7 was PCRamplified from genomic DNA isolated from Lasiodiplodia theobromae andcloned into the expression vector pCaHj505 (WO2013029496).

The final expression plasmid was transformed into the Aspergillus oryzaeM13568 expression host. A. oryzae MT3568 is a derivative of A. oryzaeJaL355 (WO02/40694) in which pyrG auxotrophy was restored by disruptingthe A. oryzae acetamidase (amdS) gene with the pyrG gene. Thecarboxypeptidase gene was integrated by homologous recombination intothe A. oryzae MT3568 host cell genome upon transformation.

The gene coding for amdS was used as marker. Transformants were selectedon pyrG media agar supplemented with 10 mM acetamide. One recombinantAspergillus oryzae clone containing the carboxypeptidase expressionconstruct was selected and was cultivated on a rotary shaking table in 42-liter baffled Erlenmeyer flasks each containing 400 ml YPM (1% Yeastextract, 2% Peptone and 2% Maltose). After 3 days cultivation time at30° C., enzyme containing supernatants were harvested by filtrationusing a 0.22 μm 1-liter bottle top vacuum filter (Corning Inc., Corning,N.Y., USA). The protein sequence is SEQ ID NO: 8.

Example 7

Purification:

The culture supernatant of O23GV9 was firstly precipitated with ammoniumsulfate (80% saturation), then dialyzed with 20 mM Bis-Tris at pH7.5.The solution was filtered with 0.45 um filter and then loaded into QSepharose Fast Flow column (GE Healthcare) equilibrated with 20 mMBis-Tris at pH7.5. A gradient of NaCl concentration from zero to 1M wasapplied as elution buffer, and then elution fractions and flow-throughfraction were collected separately for SDS-PAGE analysis. The fractionswith target protein were pooled together for evaluation.

Example 8

Strain

A fungal strain designated was isolated from a soil sample collectedfrom Yunnan Province, China, by dilution on PDA plates at 45° C. andthen purified by transferring a single conidium onto a YG agar plate.The strain was identified as Thermoascus aurantiacus, based on bothmorphological characteristics and ITS rDNA sequence.

Media

PDA medium was composed of 39 grams of potato dextrose agar anddeionized water to 1 liter.

YPG medium contained 0.4% of yeast extract, 0.1% of KH₂PO₄, 0.05% ofMgSO₄.7H₂O, 1.5% glucose in deionized water.

YPM medium contained 1% yeast extract, 2% of peptone, and 2% of maltosein deionized water.

Genomic DNA extraction of Thermoascus aurantiacus

Thermoascus aurantiacus strain was inoculated onto a PDA plate andincubated for 3 days at 45° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 ml of YPGmedium. The flasks were incubated for 3 days at 45° C. with shaking at160 rpm. The mycelia were collected by filtration through MIRACLOTH®)and frozen in liquid nitrogen. Frozen mycelia were ground, by a mortarand a pestle, to a fine powder, and genomic DNA was isolated using aDNeasy® Plant Maxi Kit following the manufacturer's instructions.

Genome Sequencing, Assembly and Annotation

The extracted genomic DNA samples were delivered to Beijing GenomeInstitute (BGI, Shenzhen, China) for genome sequencing using anILLUMINA® GA2 System (Illumina, Inc., San Diego, Calif., USA). The rawreads were assembled at BGI using program SOAPdenovo (Li et al., 2010,Genome Research 20(2): 265-72). The assembled sequences were analyzedusing standard bioinformatics methods for gene finding and functionalprediction. GeneID (Parra et al., 2000, Genome Research 10(4): 511-515)was used for gene prediction. Blastall version 2.2.10 (Altschul et al.,1990, J. Mol. Biol. 215 (3): 403-410, National Center for BiotechnologyInformation (NCBI), Bethesda, Md., USA) and HMMER version 2.1.1(National Center for Biotechnology Information (NCBI), Bethesda, Md.,USA) were used to predict function based on structural homology. Thecarboxypeptidase gene, S10_Ta, was identified directly by analysis ofthe Blast results. The Agene program (Munch and Krogh, 2006, BMCBioinformatics 7: 263) and SignalP program (Nielsen et al., 1997,Protein Engineering 10: 1-6) were used to identify start codons. TheSignalP program was further used to predict signal peptides. Pepstats(Rice et al., 2000, Trends Genet. 16(6): 276-277) was used to predictthe isoelectric points and molecular weights of the deduced amino acidsequences.

Cloning of the Carboxypeptidase Genes of Thermoascus aurantiacus fromGenomic DNA

The expression vector pCaHj505 was used for gene cloning. It containedthe TAKA-amylase promoter derived from Aspergillus oryzae and theAspergillus niger glucoamylase terminator elements. Furthermore pCaHj505had pUC18 derived sequences for selection and propagation in E. coli,and an amdS gene, which encoded an acetoamidase gene derived fromAspergillus nidulans for selection of an amds⁺ Aspergillus transformant.

The carboxypeptidase gene, S10_Ta, SEQ ID NO: 9 for the genomic DNAsequence and SEQ ID NO: 10 for the deduced amino acid sequence, wasselected for expression cloning.

Based on the DNA information obtained from genome sequencing,oligonucleotide primers, shown below, were designed to amplify thecoding sequence of the carboxypeptdase genes from the genomic DNA ofThermoascus aurantiacus NN044936. The primers were synthesized byInvitrogen, Beijing, China.

Primer1: [SEQ ID NO: 19]5′ ACACAACTGGGGATC CACC atgttgggctacgggctgttg 3′ Primer2:[SEQ ID NO: 20] 5′ CCCTCTAGATCTCGAG ggaatggcatcagatcagatcaga 3′

Lowercase characters of the forward primer represent the coding regionof the gene and lowercase characters of the reverse primer represent theflanking region of the gene, while captalized characters represent aregion homologous to insertion sites of pCaHj505 (WO2013029496). The 4underlined characters ahead of the coding sequence in the forward primerrepresent the Kozak sequence as the initiation of translation process.

Ten picomoles of the forward and reverse primers above, primer1 andprimer2 were used in a PCR reaction for amplification of the Thermoascusaurantiacus carboxypeptidase gene S10_Ta. The PCR reaction was composedof 2 μl of genomic Thermoascus aurantiacus, 10 μl of 5× Phusion® HFBuffer (Finnzyrnes Oy, Espoo, Finland), 1.5 μl of DMSO, 1.5 ul of 2.5 mMeach of dATP, dTTP, dGTP, and dCTP, and 0.6 unit of PHUSION™ HighFidelity DNA Polymerase (Finnzymes Oy, Espoo, Finland) in a final volumeof 50 μl. The amplification was performed using a Peltier Thermal Cycler(MJ Research Inc., South San Francisco, Calif., USA) programmed fordenaturing at 98° C. for 1 minute; 7 cycles of denaturing each at 98° C.for 30 seconds, annealing at 65° C. for 30 seconds with 1° C. decreaseper cycle, and elongation at 72° C. for 2 minutes; 25 cycles each at 94°C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 2 minutes; anda final extension at 72° C. for 5 minutes. The heat block then went to a4° C. soak cycle.

The PCR product was isolated by 1.0% agarose gel electrophoresis using90 mM Trisborate and 1 mM EDTA (TBE) buffer where a single product bandof approximately 2.3 kb from the reaction was visualized under UV light.The PCR product was then purified from solution by using an Illustra™GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare,Buckinghamshire, UK) according to the manufacturer's instructions.

Plasmid pCaHj505 was digested with Bam HI and Xho I, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

In-Fusion® HD Cloning Kit (Clontech Laboratories. Inc., Mountain View,Calif., USA) was used to clone the PCR fragment directly into theexpression vector pCaHj505, without the need for restriction digestion,

The purified PCR fragment and the digested vector were ligated togetherusing the In-Fusion® HD Cloning Kit according to the manufacturer'sinstructions resulting in plasmid p505-S10_Ta, in which thetranscription of the carboxypeptidase polypeptide coding sequence wasunder the control of an Aspergillus oryzae alpha-amylase gene promoter.In brief, 0.8 ul of 30 ng/ul of pCaHj505, digested with Barn HI and XhoI, and 3.2 ul of the purified PCR fragment containing ˜60 ng of theThermoascus aurantiacus carboxypeptidase gene PCR fragment were added to1 ul of 5× In-Fusion® HD Enzyme Premix. The reaction was incubated at50° C. for 15 minutes. The ligation reaction was used to transform E.coli TOP10 competent cells (TIANGEN Biotech Co. Ltd., Beijing, China).E. coli transformants containing an expression construct were detectedby colony PCR. Colony PCR is a method for quick screening of plasmidinserts directly from E. coli colonies. Briefly, a single colony wastransferred to a premixed PCR solution in a PCR tube, including PCRbuffer, MgCl₂, dNTPs, Taq DNA polymerase and primer pairs from which thePCR fragment was generated. Several colonies were screened. After thePCR, reactions were analyzed by 1.0% agarose gel electrophoresis usingTBE buffer. Plasmid DNA was prepared using a QIAPREP® Spin Miniprep Kit(QIAGEN GmbH, Hilden, Germany) from the colony showing an insert withthe expected size. The carboxypeptidase gene coding sequence inserted inp505-S10_Ta was confirmed by DNA sequencing using 3730XL DNA Analyzers(Applied Biosystems Inc, Foster City, Calif., USA).

Expression of the Thermoascus aurantiacus Carboxypeptidase Gene inAspergillus oryzae

Aspergillus oryzae strain MT3568 was used for heterologous expression ofthe gene encoding the Thermoascus aurantiacus carboxypeptidase gene. A.oryzae MT3568 is an amdS (acetamidase) disrupted derivative of A. oryzaeJaL355 (WO02/40694) in which pyrG auxotrophy was restored by disruptingthe A. oryzae acetamidase (amdS) gene with the pyrG gene.

Protoplasts were prepared according to the method described as“Transformation of Aspergillus Expression Host” in Example 2 ofUS20140179588 A1 Three μg of p505-S10_Ta were used to transformAspergillus oryzae MT3568.

The transformation of Aspergillus oryzae MT3568 with p505-S10_Ta yieldedabout 10 transformants. Four transformants were isolated to plate forreisolation and were then inoculated separately into 3 ml of YPM mediumin 24-well plate and incubated at 30° C., 150 rpm. After 3 daysincubation, 20 μl of supernatant from each culture were analyzed onNUPAGE® NOVEX® 4-12% Bis-Tris Gel with 50 mM MES (InvitrogenCorporation, Carlsbad, Calif., USA) according to the manufacturer'sinstructions. The resulting gel was stained with INSTANTBLUE® (ExpedeonLtd., Babraham Cambridge, UK). SDS-PAGE profiles of the cultures showedthat the majority of the transformants had a smear band of approximately65 kDa. The expression strain was designated as O13U8S.

Fermentation of Aspergillus oryzae Expression Strain O13U8S.

A slant of the expression strain 013U8S, was washed with 10 ml of YPMand inoculated into 4 2-liter flasks each containing 400 ml of YPMmedium, shaking at 30 C, 80 rpm. The culture was harvested on day 3 andfiltered using a 0.45 μm DURAPORE Membrane (Millipore, Bedford, Mass.,USA).

Example 9

Purification of Recombinant Carboxypeptidase by Hydrophobic InteractionChromatography (HIC)

The culture broth harvested in example 8 was precipitated with ammoniumsulfate (80% saturated). Precipitates were re-dissolved in 80 ml of 20mM PBS pH 6.0, and ammonium sulfate was replenished to get finalconcentration 1.2 M. Crude protein solution was filtered through a 0.45μm filter, and then applied to a 50 ml self-packed Phenyl Sepharose 6Fast Flow (low sub) column (GE Healthcare, Buckinghamshire, UK)equilibrated with 20 mM PBS pH 6.0 and 1.2 M ammonium sulfate buffer.Proteins were eluted with a linear 1.2 M-0 M ammonium sulfate gradient.Fractions were analyzed by SDS-PAGE using a Mini-PROTEAN TGX Stain-Free4-15% Precast Gel (Bio-Rad Laboratories, CA, United States).Carboxypeptidase activities of fractions were assessed by halo zoneassay on skim-milk agarose plate at pH 5.0, 50° C. Fractions were pooledcontaining recombinant protein bands and showing positive activities.Then the pooled solution was concentrated by ultrafiltration.

Example 10

Testing the Carboxypeptidase Specificity According to Assay I and II

The activity of carboxypeptidases of SEQ ID NO: 2, 4, 6, 8 and 10 weretested in Assay I and II together with two benchmark carboxypeptidases,i.e. CP1 (SEQ ID NO: 15) and CPY (SEQ ID NO: 14) from A. oryzae. Resultsare given in the below table 2. Calculated values for ACHA, ACLA andPro/ACHA*100 are given in table 3.

TABLE 2 Activity of different carboxypeptidases as measured in Assay Iand Assay II SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 2 NO:4 NO: 6 NO: 10 NO: 8 NO: 14 NO: 15 Pro 0.35 0.18 0.22 0.04 0.01 0.130.02 Ala 0.46 0.33 0.39 0.05 0.01 0.28 0.12 Val 0.57 0.56 0.57 0.09 0.030.55 0.22 Leu 0.59 0.61 0.57 0.04 0.03 0.60 0.21 Ile 0.63 0.61 0.59 0.090.02 0.57 0.25 Met 0.59 0.57 0.58 0.08 0.03 0.54 0.21 Phe 0.60 0.59 0.590.03 0.02 0.56 0.16 Trp 0.49 0.40 0.44 0.06 0.02 0.38 0.15 Gly 0.05 0.030.03 0.01 0.00 0.01 0.01 Ser 0.15 0.07 0.09 0.04 0.01 0.05 0.08 Tyr 0.440.44 0.44 0.05 0.03 0.31 0.12 Asn 0.14 0.06 0.07 0.05 0.00 0.04 0.09 Gln0.00 0.04 0.02 0.01 0.05 0.07 0.01 Asp 0.04 0.01 0.02 0.01 0.00 0.010.01 Glu 0.02 0.01 0.01 0.01 0.00 0.01 0.02 His 0.08 0.05 0.06 0.06 0.000.02 0.07 Lys 0.00 0.01 0.00 0.22 0.24 0.11 0.32 Arg 0.03 0.01 0.01 0.230.14 0.02 0.23

The result show that the carboxypeptidases of SEQ ID NO: 2, 4 and 6 allhave an activity of at least 0.15 on Proline, whereas the prior artcarboxypeptidase CPY has an activity of 0.13.

TABLE 3 Average activity on hydrophobic amino acids excluding Pro (ACHA)and relative activity on Pro compared to average activity on hydrophobicamino acids excluding Pro (Pro/ACHA*100). Average activity on lys, Arg(ACLA) and relative activity on Lys, Arg compared to average activity onhydrophobic amino acids excluding Pro (ACLA/ACHA). SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID NO: 2 NO: 4 NO: 6 NO: 10 NO: 8 NO: 14 NO: 15ACHA 0.56 0.52 0.53 0.06 0.02 0.50 0.19 Pro/ACHA *100 61 35 42 58 53 2511 ACLA 0.01 0.01 0.00 0.23 0.19 0.06 0.28 ACLA/ACHA*100 2 2 0 364 85412 146

As seen in the table the new carboxypeptidases are all characterised byhaving a higher ratio of activity on Pro versus average activity onhydrolphobic amino acids (Pro/ACHA*100) compared to the benchmarkcarboxypeptidases (SEQ ID NO: 14 and 15).

Example 11 Testing of Carboxy-Peptidases in Soy and Gluten on Top ofAlcalase and Flavourzyme in Small Scale

The carboxy-peptidases (exo-peptidases) were screened on top of Alcalase(Alc) and Flavourzyme (FZ using a 6% protein suspension of either soybean meal or wheat gluten. Incubation was done in a Deep Well Plate(DWP) with shaking overnight. After incubation the samples were dilutedin MQ water and analysed using the OPA assay (Assay V).

Substrate:

Soy bean meal: Protein content: 52% w/w on soy bean meal

Wheat gluten: Protein content: 73.3% w/w on wheat gluten

Enzymes and Dosages:

Alcalase AF 2.4 L: 0.4% on protein

Flavourzyme 1000 L: 0.8% on protein

Exo-peptidase candidate: 1.5-2 mg enzyrneprotein/g protein

DWP: 1 ml DPW, wide-bottomed to ensure mixing; incubation in EppendorfThermomixer

Incubation:

Temperature: 55° C.

pH as is: 6.0-6.5

Shaking: 1200 rpm

Time: overnight, 18-19 hrs

Set-Up:

DPW assay: Suspensions of the substrates were made giving a finalprotein concentration of 6.5% based on the protein content given above,and considering the further dilution of the substrate when enzymes wereadded. Total volume in the wells were 285 ul. All enzymes were dilutedas appropriate before addition. Substrates were pretreated usingAlcalase and Flavourzyme to ensure that dispensing into the wells werepossible. Alcalase and Flavourzyme were therefore added to a largerportion of the substrate and the mixture incubated at 55′C for 50-60min. 245 ul substrate per well was then transferred to the MWP and 40 ulof the exo-peptidase added.

The outer column and row of the deep well plate were not used due totemperature fluctuations. Samples were run in quadruplicate and a sampleholding Alcalase and Flavourzyme included as a reference. Plates wereleft overnight in a Thermomixer. After incubation the samples werediluted in MQ water (e.g. 15 ul in 985 ul, DWP) and the degree ofhydrolysis determined using an OPA assay (Assay V). Samples wereanalysed within 20 min, since no inactivation of the samples were done.

Results are given in the tables 4-5 below. DH of the sample having onlyAlcalase and Flavourzyme were set as 100% and the response of thesamples with exo-peptidase on top of Alcalase and Flavourzyme comparedto this. Benchmark enzymes included were SEQ ID NO: 14 acarboxypeptidase from A. oryzae with preference for hydrophobic aminoacids and SEQ ID NO: 15 a carboxypeptidase from A. oryzae withpreference for basic amino acids but also activity on hydrophobic aminoacids.

As seen the top candidate in both soy and gluten were enzyme sample SEQID NO: 2, characterised as a carboxypeptidase with a high activity onhydrophobic amino acids in general, and with a high activity on Pro inparticular. Table 1: Final DH obtained in soy hydrolysates relative tothe DH of the sample with background enzymes (Alcalase+Flavourzyrne) setto 100. Dosages of exo-candidates were 2 mg enzyme protein/g proteinexcept for SEQ ID NO: 4 and SEQ ID NO: 6 where dosages were 1.5 and 1.8mg ep/g protein respectively.

TABLE 4 Enzyme ID DH/DH_(Alc+FZ) *100 SEQ ID NO: 2 200 SEQ ID NO: 4 169SEQ ID NO: 10 166 SEQ ID NO: 6 160 SEQ ID NO: 8 151 SEQ ID NO: 14 = CPY141 SEQ ID NO: 15 = CP1 138

TABLE 5 Final DH obtained in gluten hydrolysates relative to the DH ofthe sample with background enzymes (Alcalase + Flavourzyme) set to 100.Dosages of exo-candidates were 2 mg enzyme protein/g protein except forSEQ ID NO: 6 where 1.8 mg ep/g protein was used. Enzyme IDDH/DH_(Alc+FZ) *100 SEQ ID NO: 2 157 SEQ ID NO: 10 154 SEQ ID NO: 6 150SEQ ID NO: 15 = CP1 151 SEQ ID NO: 4 144 SEQ ID NO: 8 136 SEQ ID NO: 14= CPY 120

Example 12

Testing of carboxy-peptidase in soy on top of Alcalase

Further small scale tests in the DWP set-up were done combiningcarboxy-peptidase and aminopeptidase (AP2) in soy and using either 0.5%Alcalase and 1% Flavourzyme as a background or 0.5% Alcalase alone.Dosage of exo-peptidase were 1 mg ep/g protein both when used alone orin combinations. Results are shown in table 6.

When using single candidates, results showed that SEQ ID NO: 2 wasclearly superior giving the highest DH both when combined with Alcalasealone (DH 37) or with Alcalase and Flavourzyme (DH 46). When added ontop of Alcalase only, the performance of SEQ ID NO: 2 was better thanwhen using 1% FZ (37 vs. 28) on top of Alcalase. Performance of SEQ IDNO: 15 and SEQ ID NO: 16 were comparable both when added on top ofAlc+Fz and when used with Alc only. The combined treatment showed thehighest final DH value.

TABLE 6 DH obtained when hydrolysing soy protein with best candidates.Background was 0.5% Alc +/− 1% FZ. Candidates were dosed 1 mg ep/gprotein in single treatments and 1 + 1 mg ep/g protein in combinedtreatment. Alcalase Alcalase + Flavourzyme Background 14 28 +SEQ ID NO:16 26 37 +SEQ ID NO: 15 27 37 +SEQ ID NO: 2 37 46 +SEQ ID NO: 2 + 54 55SEQ ID NO: 16 + SEQ ID NO: 15

Example 13: Testing of Best Candidate in Dose Response in Soy or Glutenon Top of Alcalase and Flavourzyme

Dose response of the best candidate, carboxypeptidase SEQ ID NO: 2 wasdone in soy on a background of 0.8% Alcalase+1.6% Flavourzyme and ingluten on a background of 0.4% Alcalase+0.8% Flavourzyme. Test were runin the DWP set-up. Results are given in table www and show a significanteffect on DH also at the lowest dosages.

TABLE 7 % DH obtained when hydrolysing soy or gluten with bestexo-peptidase in increasing dosage. Mg ep/g protein of carboxypeptidaseSoy Gluten 0 (=Background) 23 31 +0.5 40 46 +1.0 42 48 +1.5 44 47 +2.046 49

Example 14: Testing of Carboxy-Peptidases in Soy and Gluten on Top ofAlcalase and Flavourzyme in 25 g Scale

25 g scale: Substrate suspensions were prepared as above and Alcalaseand Flavourzyme added for pretreatment. Compared to the DWP assaydosages of Alcalase and FZ was doubled (0.8% and 1.6%). After incubationfor 50 min at 55° C., 21.5 g substrate was transferred to 50 nil bluecap flasks and 3.5 g of the diluted exo-peptidase added (1 mg enzymeprotein/g protein). Combinations of a carboxypeptidase and anaminopeptidase were also tested. Dosages were 1+1 mg ep/g protein.Flasks were held in a heated water bath (55° C.) with magnetic stirring.After overnight incubation (20 hrs), 1.8 ml samples were withdrawn andenzymes inactivated in a Thermomixer at 95° C. for 10 min. Samples werediluted and analysed using OPA assay (Assay V).

Results are given in table 8 and show that the best performing candidate(SEQ ID NO: 2) identified in the smaller scale assay (DWP) also provedsuperior in larger scale.

TABLE 8 DH obtained in 25 g scale when hydrolysing soy or gluten proteinwith best candidates. Background was 0.8% Alc + 1.6% FZ. Candidates weredosed 1 mg ep/g protein in single treatments and 1 + 1 mg ep/g proteinin combined treatments. Soy Gluten DH Delta DH DH Delta DH Background 3745 +SEQ ID NO: 16 46 9 48 3 +SEQ ID NO: 15 47 10 53 8 +SEQ ID NO: 2 5316 53 8 +SEQ ID NO: 2 + 65 28 58 13 SEQ ID NO: 16 +SEQ ID NO: 15 + 55 1858 13 SEQ ID NO: 16

Background or reference DH obtained in the larger scale assay wasclearly higher than in the DWP assay, which could be expected since thebackground dosages of Alcalase and FZ were higher. In addition to higherenzyme dosage improved mixing in larger scale might also increasehydrolysis.

Looking at the effect of the single candidates in the 25 g scaleexperiment and the combined treatments using one carboxypeptidase+oneaminopeptidase, overall effects on DH seemed to be more or lessadditive.

Amino Acids Analysis

Results of analysing the amino acid composition of the hydrolysates areshown in table 9.

TABLE 9 Amino acids in soy or gluten hydrolysates using the bestcandidates on a background of 0.8% Alcalase and 1.6% Flavourzyme. Dataare mg/L amino acids and summed per amino acid characteristics. Groupsare: Hydrophobic amino acids consisting of: Ala, Val, Met, Trp, Phe,Ile, & Leu; Pro is given separately; Polar uncharged amino acidsconsisting of: Asn, Ser, Gln, Thr, Tyr & His/Gly (His/Gly could not beseparated on the column so His is included in the polar uncharged group,even though it is a basic amino acid); Acidic amino acids consisting of:Glu & Asp; and Basic amino acids consisting of: Lys & Arg. Cys was notanalysed. Amino acids, mg/L Polar Hydrophobic Pro uncharged + His AcidicBasic Sum Soy Background: 7399 230 5104 2171 1455 16359 +SEQ ID NO: 169426 239 8435 4200 4620 26921 +SEQ ID NO: 15 11023 816 8864 3967 525329922 +SEQ ID NO: 2 14038 957 9722 3417 4835 32968 +SEQ ID NO: 2 + SEQID NO: 16 15433 790 12706 5254 6061 40245 +SEQ ID NO: 15 + SEQ ID NO: 1612820 642 11711 5957 5587 36717 Gluten Background 11001 283 14657 23942347 30682 +SEQ ID NO: 16 11714 647 17294 2848 2656 35159 +SEQ ID NO: 1512756 2174 18116 2704 2538 38297 +SEQ ID NO: 2 14726 2580 18518 27242833 41381 +SEQ ID NO: 2 + SEQ ID NO: 16 15629 3385 20557 2771 241244454 +SEQ ID NO: 15 + SEQ ID NO: 16 14540 3025 22259 4072 2830 46726

As seen in the table the sum of free amino acids increased when theexo-peptidases were added on top of Alcalase and Flavourzyme. Bestperformance was achieved when combining carboxy, and aminopeptidases. Ofthe single candidates SEQ ID NO: 2 was superior, showing 100% increasein total amount of liberated amino acids in soy and 35% in gluten.

Example 15

Testing of Best Candidates at Higher Protein Concentration on Top ofAlcalase

A wheat gluten suspension was made having a final protein concentrationof 10% based on a protein content of 73.3% w/w as is in the vital wheatgluten and considering that the substrate was diluted further whenenzymes were added, Hydrolysis was carried out in 2 ml Eppendorf tubesholding 1.8 ml substrate, incubated in an Eppendorf thermomixer withshaking (1200 rpm). Duplicate determinations were made. Incubationtemperature was 55° C., and incubation time 18 hours. pH was notadjusted. Enzymes were inactivated by holding the samples at 90° C. for10 min, and % DH (OPA analysis) and free amino acids (HPLC) wereanalyzed.

Amino Acids Analysis

Results of analysing the amino acid composition of the hydrolysate areshown in table ccc.

TABLE 10 DH (OPA assay V) and amino acids in gluten hydrolysates usingthe best candidates on a background of 1% Alcalase. Dosage of candidateswere 0.5 mg ep/g protein and 0.5 + 0.5 mg ep/g protein in the combinedtreatment. Data are mg/L amino acids and are summed per amino acidcharacteristics. Groups are: Hydrophobic amino acids consisting of: Ala,Val, Met, Trp, Phe, Ile, & Leu; Pro is given separately; Polar unchargedamino acids consisting of: Asn, Ser, Gln, Thr, Tyr & His/Gly (His/Glycould not be separated on the column so His is included in the polaruncharged group, even though it is a basic amino acid); Acidic aminoacids consisting of: Glu & Asp; and Basic amino acids consisting of: Lys& Arg. Cys was not analysed. mg/L % Polar DH Hydrophobic Pro uncharged +His Acidic Basic Sum Background 9 1433 35 1061 235 351 3125 +SEQ ID NO:15 21 6734 1569 9103 873 858 19137 +SEQ ID NO: 2 30 11379 2323 9122 819934 24576 +SEQ ID NO: 8 25 7663 378 9813 1330 1753 20938 +SEQ ID NO: 626 10083 1579 7694 685 841 20882 +SEQ ID NO: 16 32 8076 38 13539 12511552 24455 +SEQ ID NO: 15 + SEQ ID NO: 16 40 11506 930 19233 1958 188435511

As seen in the table, the sum of free amino acids increasedsignificantly when the exo-peptidases were added on top of Alcalase.Best performance was achieved when combining carboxy, andaminopeptidases. Of the single candidates SEQ ID NO: 2 was superior,showing the highest amount of liberated amino acids, followed by SEQ IDNO: 16. For the carboxypeptidases (SEQ ID NO: 2 and SEQ ID NO: 6)especially levels of hydrophobic amino acids and Pro were high, whilefor the aminopeptidase (SEQ ID NO: 16) levels of polar uncharged, acidicand basic amino acids were high.

Example 16

Testing of best candidates in combination with aminopeptidase (SEQ IDNO: 16) at higher protein concentration on top of Alcalase

Testing of the different carboxypetidases combined with aminopeptidasewere done in a 10% wheat gluten protein suspension. The set-up was thesame as the one used in Example 15.

TABLE 11 DH (OPA analysis) and amino acids in gluten hydrolysates usingthe best candidates combined with aminopeptidase on a background of 1%Alcalase. Dosage of carboxypeptidases and of aminopeptidase were: 0.5 mgep/g protein. Data are mg/L amino acids and are summed per amino acidcharacteristics. Groups are: Hydrophobic amino acids consisting of: Ala,Val, Met, Trp, Phe, Ile, & Leu; Pro is given separately; Polar unchargedamino acids consisting of: Asn, Ser, Gln, Thr, Tyr & His/Gly (His/Glycould not be separated on the column so His is included in the polaruncharged group, even though this is a basic amino acid); Acidic aminoacids consisting of: Glu & Asp; and Basic amino acids consisting of: Lys& Arg. Cys was not analysed. mg/L % Polar DH Hydrophobic Pro uncharged +His Acidic Basic Sum Background 10 1527 46 1119 241 382 3315 +SEQ ID NO:15 + SEQ ID NO: 16 48 13470 1038 21767 2087 1931 40293 +SEQ ID NO: 2 +SEQ ID NO: 16 57 16649 2157 21379 2201 2218 44607 +SEQ ID NO: 10 + SEQID NO: 16 48 13525 945 21900 2112 1911 40392 +SEQ ID NO: 6 + SEQ ID NO:16 54 17883 1562 21490 2189 2398 45522

As seen in the table the sum of free amino acids increased significantlywhen the combination of carboxy-peptidases and aminopeptidase were addedon top of alcalase. Best performance was achieved with thecarboxypeptidases SEQ ID NO: 2 and SEQ ID NO: 6. Especially levels ofhydrophobic amino acids and pro were high in these samples.

Example 17

Testing of Carboxy-Peptidases in Whey in Combination with Alcalase

Performance of the carboxy-peptidase (SEQ ID: 2) was compared to thebenchmark enzymes CPY and CP1 (SEQ ID 14 and SEQ ID 15) when combinedwith Alcalase in a 6% whey protein solution at pH 7.5.

Substrate:

Whey Protein Isolate: Protein content: 90% w/w

Enzymes and Dosages:

Alcalase AF 2.4 L: 0.4% w/w on protein

Exo-peptidase candidate: 0.05-0.2 mg enzymeprotein/g protein

Incubation:

Temperature: 55° C.

pH: 7.5

Time: 4 hrs

Set-up

Incubation was done in 50 ml glass flasks with stirring. Afterincubation pH was readjusted to 7.5 using NaOH and enzymes inactivatedby holding the samples at 95° C. for 15 min (total time includingheat-up). Samples were then diluted in MQ water and analysed using anOPA assay (Assay V).

Results are given in the table 12 below. DH of the sample with Alcalasealone was 14. Dosing of the carboxypeptidases were adjusted to give afinal DH of approximately 20 in all samples. As seen, performance of SEQID: 2 was clearly superior, when comparing on enzyme protein dosagelevel, giving a final DH of 20.3 at a dosage of 0.05 mg enzyme protein/gprotein. The benchmark enzymes had to be dosed at 0.1 and 0.2 mg ep/g,respectively, for a similar performance. This clearly illustrates thebetter performance and higher specific activity of the SEQ ID: 2carboxypeptidase.

TABLE 12 Final DH obtained in whey protein hydrolysates. Dosage ofAlcalase was 0.4%, and of exo-peptidases between 0.05 and 0.2 mg enzymeprotein/g protein. Enzyme ID DH Alcalase 0.4% 14 Alcalase 0.4% + SEQ ID:14, 0.1 mg ep/g 20.5 Alcalase 0.4% + SEQ ID: 15, 0.2 mg ep/g 19.2Alcalase 0.4% + SEQ ID: 2, 0.05 mg ep/g 20.3 Alcalase 0.4% + SEQ ID: 2,0.06 mg ep/g 21.8 Alcalase 0.4% + SEQ ID: 2, 0.1 mg ep/g 23.5 Alcalase0.4% + SEQ ID: 2, 0.2 mg ep/g 27.1

1. A method for producing a protein hydrolysate comprising: a. providingan aqueous solution or suspension of a protein substrate; and b.exposing the aqueous solution or suspension of the protein substrate toa polypeptide having endopeptidase activity and to a polypeptide havingcarboxypeptidase activity, to obtain the protein hydrolysate; whereinthe polypeptide having carboxypeptiddase activity is characterised byhaving a Pro/ACHA*100 ratio of at least
 30. 2. The method according toclaim 1, wherein the concentration of the protein substrate in theaqueous solution or suspension is in the range of 5-35%.
 3. The methodaccording to claim 1, wherein the polypeptide having carboxypeptidaseactivity is selected from the group consisting of a. a polypeptidehaving at least 60% sequence identity to the mature polypeptide of SEQID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10; b.a polypeptide encoded by a polynucleotide having at least 60% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQID NO: 3 or SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or the cDNAsequence thereof; c. a variant of the mature polypeptide of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 comprisinga substitution, deletion, and/or insertion at one or more positions; andd. a fragment of the polypeptide of (a), (b) or (c) that hascarboxypeptidase activity.
 4. An isolated polypeptide havingcarboxypeptidase activity, selected from the group consisting of: a. apolypeptide having at least 60% sequence identity to the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10; b. a polypeptide encoded by a polynucleotide having atleast 60% sequence identity to the mature polypeptide coding sequence ofSEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9or the cDNA sequence thereof; c. a variant of the mature polypeptide ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10comprising a substitution, deletion, and/or insertion at one or morepositions; and d. a fragment of the polypeptide of (a), (b) or (c) thathas carboxypeptidase activity.
 5. A liquid or granulate compositioncomprising the polypeptide of claim
 4. 6. A whole broth formulation orcell culture composition comprising the polypeptide of claim
 4. 7. Apolynucleotide encoding the polypeptide of claim
 4. 8. A nucleic acidconstruct or expression vector comprising the polynucleotide of claim 7operably linked to one or more control sequences that direct theproduction of the polypeptide in an expression host.
 9. A recombinanthost cell comprising the polynucleotide of claim 7 operably linked toone or more control sequences that direct the production of thepolypeptide. 10-11. (canceled)
 12. A method for producing a polypeptidehaving carboxypeptidase activity, comprising cultivating the host cellof claim 9 under conditions conducive for production of the polypeptide.13. The method according to claim 12, further comprising recovering thepolypeptide.
 14. (canceled)
 15. Protein hydrolysate comprising freeamino acids, wherein the amount of free amino acids is at least 20 g per100 gram protein, the total amount of Ala, Val, Ile, Leu, Met, Phe andTrp is at least 7 g per 100 g protein and at least 1.0 g Pro per 100 gprotein.